The GRAS transcription factor CsTL regulates tendril formation in cucumber

Abstract

Cucumber (Cucumis sativus, Cs) tendrils are slender vegetative organs that typically require manual removal to ensure orderly growth during greenhouse cultivation. Here, we identified cucumber tendril-less (tl), a Tnt1 retrotransposon-induced insertion mutant lacking tendrils. Map-based cloning identified the mutated gene, CsaV3_3G003590, which we designated as CsTL, which is homologous to Arabidopsis thaliana LATERAL SUPPRESSOR (AtLAS). Knocking out CsTL repressed tendril formation but did not affect branch initiation, whereas overexpression (OE) of CsTL resulted in the formation of two or more tendrils in one leaf axil. Although expression of two cucumber genes regulating tendril formation, Tendril (CsTEN) and Unusual Floral Organs (CsUFO), was significantly decreased in CsTL knockout lines, these two genes were not direct downstream targets of CsTL. Instead, CsTL physically interacted with CsTEN, an interaction that further enhanced CsTEN-mediated expression of CsUFO. In Arabidopsis, the CsTL homolog AtLAS acts upstream of REVOLUTA (REV) to regulate branch initiation. Knocking out cucumber CsREV inhibited branch formation without affecting tendril initiation. Furthermore, genomic regions containing CsTL and AtLAS were not syntenic between the cucumber and Arabidopsis genomes, whereas REV orthologs were found on a shared syntenic block. Our results revealed not only that cucumber CsTL possesses a divergent function in promoting tendril formation but also that CsREV retains its conserved function in shoot branching.

Figure 1.

Fine mapping and expression analysis of CsTL, a novel gene controlling tendril formation in cucumber. Growth habit of WT plants (A) and tl mutant plants (B) 2 months after sowing. Boxplots depicting the distribution of tendril number (C) and branch number (D) in both 2.5-month-old WT and tl plants (n = 9). Significance analyses were performed with two-tailed Student's t-test (**P < 0.01). E) and F) Preliminary mapping of tl by using BSA-seq. E) Identification of the candidate interval for the tl gene was achieved through the SNP index association analysis method. The tl locus is situated within a 3.73-Mbp interval on Chromosome 3. The abscissa represents the chromosome name, while the ordinate denotes the SNP index value. F) Results of BSA-seq mapping of the tendril-regulating gene based on the Euclidean distance (ED) algorithm. The abscissa is the chromosome name, and the ordinate is the ED value. The colored dots represent the ED values of each SNP site. The black line represents the fitted ED value. The red line represents the correlation threshold. G) and H) Map-based cloning of genes in the tl locus. G) Four Tnt1 insertion sites within the candidate region used for designing markers for genotyping a total of 340 plants in the F₂ population. H) Annotated genes in the final interval. CsaV3_3G003590 is cucumber TL candidate. I) Expression analysis of CsTL in different cucumber organs. Error bars represent mean ±SD. Values are means ± SD (n = 3). Significance was determined using one-way ANOVA (P < 0.05). J) to L) In situ hybridization analysis of CsTL expression in SAM tissue. The sense probes of CsTL were hybridized as controls (CK) (J). The black arrows in K and L indicate the mRNA signals. Scale bars = 50 μm.

Figure 2.

Functional analysis of CsTL by CRISPR/Cas9-engineering and OE system. A) sgRNA target sites in the first exon of CsTL and editing results in cucumber determined by Sanger sequencing. The two target sites of the sgRNAs and adjacent sequences in the first exon are shown for both the WT plant and the gene-edited mutants. Deleted nucleotides are denoted by dashes. The sgRNA sequences are highlighted in red, and the PAM sites are marked in blue. Genotypic analysis of CsTL knockout plants with the CU2 background revealed partial deletions in the DNA sequence of the first exon. B) to D) Comparison of phenotypic differences between WT, CsTL^CR-1, and CsTL^CR-3 knockout lines. LB, lateral branch; T, tendril. Scale bars = 5 cm. E) Diagram illustrating the positioning of tendrils and branches at the initial 20 nodes in the WT and CsTL mutant plants. Each layer represents a node in a cucumber plant. A green square signifies a node that produces a normal tendril but no branch, while a boxed green square indicates a node that produces both a normal tendril and a branch. A yellow square represents a node that produces neither a branch nor a tendril, and a boxed yellow square represents a node that produces a branch but no tendril. Comparison of the internal composition of shoot apexes in WT plant (F), CsTL knockout plant (G), and HW plant (H) at 30 days of age using 3D micro-CT imaging. TP, tendril primordium; FP, flower primordium; LBP, lateral branch primordium. Scale bars = 300 μm. Tendril phenotypes in WT (I), OE-2 (J), and OE-3 (K) plants. Tendrils are indicated by white arrows. T, tendril. Scale bars = 5 cm.

Figure 3.

Analyses of relationships between CsTL and CsTEN and CsUFO. A) Heat map of gene expression levels of DEGs in WT and CsTL^CR lines from three independent biological replicates (n = 3). A colored legend positioned to the left of the map indicates FPKM (fragments per kilobase of transcript per million mapped reads) values. The data for the WT serve as the control (CK). RT-qPCR analyses of tendril-related genes CsTEN(B) and CsUFO(C) expression in CsTL^CR lines. Significance analyses compared with WT were performed with two-tailed Student's t-test (**P < 0.01). The values represent the mean ± SD (n = 3). Phenotypic comparison of WT (D), CsTL^CR line (E), CsTEN^CR line (F), and Csufo mutant (G). H) Yeast one-hybrid assay showing that CsTL could not bind to the promoter of either CsTEN or CsUFO. I) Schematic diagrams of the effector and reporter constructs used in the LUC reporter transient expression system. J) Effects of CsTL on the CsTEN and CsUFO promoter activity. The LUC/REN ratio represents the LUC activity relative to the internal control REN. Values are mean ± SD (n = 6).

Figure 4.

CsTL directly interacts with CsTEN to enhance CsTEN-mediated CsUFO expression. A) Yeast two-hybrid assay showed that CsTL interacts with CsTEN in yeast cells grown on SD-Leu/-Trp/-His/-Ade/X-α-gal selective medium by observation of the growth of transformants. The combinations containing either empty pGADT7 or empty pGBKT7 vectors were used as negative controls. B) In vitro GST pull-down assay further confirming the physical interaction between CsTL and CsTEN. The combination of GST and His-CsTEN was used as negative control. C) BiFC assay validating the interaction between CsTL and CsTEN in N. benthamiana epidermal leaf cells. Scale bars = 20 μm. D) Firefly LUC complementation imaging assay further showed that CsTL interacts with CsTEN. CsTL-cLUC and nLUC-CsTEN were transiently co-expressed in N. benthamiana leaves, and the remaining combinations were used as negative controls. E) Co-IP analysis showed the interaction of CsTL with CsTEN in N. benthamiana leaves. Co-IP assay was performed using an agarose conjugated anti-GFP antibody. The expression levels of CsUFO(F) and CsTEN(G) were assessed in 35S:CsTL-GR transgenic plants following treatments with DEX, CHX, and DEX + CHX, respectively. Asterisks denote significant differences between samples treated with different chemicals (**P < 0.01, Student's t-test). The values are presented as mean ± SD (n = 3). H) Firefly LUC and Renilla reniformis LUC activity assay in N. benthamiana leaves by co-expression of Pro35S:CsTL and/or Pro35S:CsTEN with ProCsUFO:LUC. The LUC/REN ratio from the empty vector (62-SK) combined with ProCsUFO:LUC was used as a calibration. Values are mean ± SD (n = 6).

Figure 5.

Role of CsREV in branch development in cucumber. A) to C) In situ hybridization analysis of CsREV expression in the shoot apex of WT (B) and CsTL mutant (C). The sense probe of CsREV was hybridized as negative control (CK) (A). AM, axillary meristem. Scale bars = 20 μm. D) Expression levels of CsREV in CsTL knockout lines. Values are mean ± SD (n = 3). E) Schematic diagram of the CsREV gene showing the two CRISPR/Cas9-targeting sites located in its first exon and the induced mutations at the first site in mutant lines 1, 2, and 3. Comparison of WT (F–H) and CsREV^CR-1 plants (I–K) from overall plant appearance (F and I) to individual leaf axils (G, H, J, and K). Scale bars = 5 cm. L) Diagram illustrating the positioning of tendrils and branches in the initial 20 nodes of both WT and CsREV^CR plants. Each layer corresponds to a node in a cucumber plant. Green squares denote nodes generating regular tendrils without branches, while boxed green squares signify nodes producing both normal tendrils and branches. Yellow squares represent nodes producing neither branches nor tendrils, with boxed yellow squares indicating nodes generating only branches without tendrils.

Figure 6.

Syntenic analyses of CsTL-containing region and CsREV-containing region between cucumber and Arabidopsis genome and within cucumber genome. A) Syntenic relationship between CsTL-containing region on Chromosome 3 of cucumber and its syntenic region on Chromosome 1 of Arabidopsis. The location of CsTL is highlighted in magenta. B) Syntenic analysis of cucumber Chromosomes 3 and 4 with the AtLAS-containing region in Arabidopsis. C) Syntenic relationship between CsREV-containing region on Chromosome 6 of cucumber and AtREV-containing region on Chromosome 5 of Arabidopsis. The location of CsREV is highlighted in green. D) Syntenic relationship between CsREV-containing region on Chromosome 6 of cucumber and another region on Chromosome 6. Gray lines indicate homologous gene pairs in the syntenic regions. The location of CsREV is highlighted in green. E) Syntenic relationship between CsTL-containing region on Chromosome 3 of cucumber and two regions on Chromosome 5 and 6. Gray lines indicate homologous gene pairs in the syntenic regions. The location of CsTL is highlighted in magenta.

Figure 7.

Model for engineering ideal plant architecture in cucumber. A) In model plant Arabidopsis, Lateral suppressor (LAS) together with its downstream gene REVOLUTA (REV) directly modulate shoot branching. B) However, our results indicate that the LAS homolog in cucumber, CsTL, regulates tendril formation by directly forming a complex with CsTEN to further enhance CsUFO expression, while CsREV conservatively functions in regulating branching in cucumber. C) Cucumber varieties cultivated in a protected environment traditionally produce fruits, tendrils, and branches in leaf axils, leading to competition for nutritional allocation among these three lateral organs. Through the editing of CsTL and/or CsREV in cucumber, it may be possible to develop cucumber varieties without tendrils, without branches, or without both tendrils and branches.