CsPHYB- CsPIF3/ 4 Regulates Hypocotyl Elongation by Coordinating the Auxin and Gibberellin Biosynthetic Pathways in Cucumber (Cucumis sativus L.)

Abstract

Hypocotyl length is closely related to quality in seedlings and is an important component of plant height vital for plant-type breeding in cucumber. However, the underlying molecular mechanisms of hypocotyl elongation are poorly understood. In this study, the endogenous hormone content of indole acetic acid (IAA) and gibberellin (GA₃) showed an increase in the long hypocotyl Csphyb (phytochrome B) mutant AM274M compared with its wild-type AM274W. An RNA-sequencing analysis identified 1130 differentially expressed genes (DEGs), of which 476 and 654 were up- and downregulated in the mutant AM274M, respectively. A KEGG enrichment analysis exhibited that these DEGs were mainly enriched in the plant hormone signal transduction pathway. The expression levels of the pivotal genes CsGA20ox-2, in the gibberellin biosynthesis pathway, and CsYUCCA8, in the auxin biosynthesis pathway, were notably elevated in the hypocotyl of the mutant AM274M, in contrast to the wild-type AM274W. Additionally, GUS staining and a dual-luciferase reporter assay corroborated that the phytochrome-interacting factors CsPIF3/4 can bind to the E(G)-box motifs present in the promoters of the CsGA20ox-2 and CsYUCCA8 genes, thereby modulating their expression and subsequently influencing hypocotyl elongation. Consequently, this research offers profound insights into the regulation of hypocotyl elongation by auxin and gibberellin in response to light signals and establishes a crucial theoretical groundwork for cultivating robust cucumber seedlings in agricultural practice.

Keywords: CsPHYB; CsPIF3/4; auxin; cucumber; gibberellin; hypocotyl elongation.

Figure 1

Phenotypic analyses of the long-hypocotyl mutant AM274M. (A) Seedling phenotypes of the long-hypocotyl mutant AM274M and its wild-type AM274W. (C) Phenotypic characteristics of AM274M, lh2, and AM274Mlh2. (B,D) The data of the mutant AM274M, wild-type AM274W, mutant lh2, and the double-mutant AM274Mlh2 hypocotyl length were collected 15 days after germination, as shown in (A,C). Scale bar = 2 cm. Data are means ± SD. “**” and the capital letters A–C all indicate statistically significant differences in t-tests at p < 0.01 between the mutant and WT.

Figure 2

Determination of hormone content in the hypocotyl of the mutant AM274M and wild-type AM274W. (A–E) ZT, GA₃, ABA, IAA, and JA content in the hypocotyl of the mutant AM274M and wild-type AM274W. Significance analysis was conducted with two-tailed Student’s t-tests (** p < 0.01) between the mutant and WT.

Figure 3

Effects of exogenous GA₃ and IAA on hypocotyl elongation in the wild-type AM274W and mutant AM274M. (A–D) Phenotype and length of hypocotyl of the wild-type (A,C) and AM274M (B,D) after exogenous GA₃ and IAA treatment. Scale bar = 2 cm; “*” and “**” denote statistically significant differences in t-tests at p < 0.05 and p < 0.01 between the mutant and WT.

Figure 4

The RNA-seq showed the impact of the AM274M mutation on gene expression. (A) The differential number of DEGs between the AM274M mutant and the AM274W wild-type. (B) DEG volcano map, where the red dots represent the upregulated genes in AM274M, the blue dots represent the downregulated genes, and the gray dots represent the non-differentially expressed genes. (C) GO enrichment analysis of differentially expressed genes between AM274M and AM274W. (D) KEGG enrichment analysis of differentially expressed genes between AM274M and AM274W.

Figure 5

The effects of wild-type AM274W and mutant AM274M on different gene expression levels on the 7th day after germination. Significance level with a two-tailed Student’s t-test (* p < 0.05 and ** p < 0.01).

Figure 6

GUS staining analysis and dual-luciferase reporter assay. (A,B) Prediction of cis-acting elements of the CsGA20ox-2 and CsYUCCA8 promoters as a schematic diagram; P1, P2, P3, and P4 represent four different E-box elements, and P5 indicates the G-box element. The black boxes represent gene exons, and the blue lines represent introns and promoters, respectively. (C) Construction of effector vectors (35S:CsPIF3/4) and reporter vectors (mini35S-GUS, 3×GA20ox2-P1/P2/P3/P4-mini35S-GUS, 3×YUCCA8-P5-mini35S-GUS). (D) GUS staining of tobacco leaves injected with a single reporter and GUS staining of tobacco leaves co-infected with the effector 35S:CsPIF3 and the reporter 3×GA20ox2-P1/P2/P3/P4/P5-mini35S-GUS, 35S:CsPIF4, and 3×YUCCA8-P5-mini35S-GUS. (E) Construction of the effector vector 35:CsPIF3/4 and the reporter vector ProGA20ox2-P1/P2/P3/P4-LUC and proYUCCA8-P5-LUC for dual-luciferase activity assay. (F–J) 62-SK+P1/P2/P3/P4/P5 means that the empty vector effector was co-injected with the reporter, respectively; 35S:PIF3+P1/P2/P3/P4 indicates that the effector 35S:PIF3 was co-injected with the reporter ProGA200x2-P1/P2/P3/P4-LUC and proYUCCA8-LUC-P5, respectively; and 35S:PIF4+P5 indicates the co-injection of the effector 35S:PIF4 and proYUCCA8-LUC-P5. “**” denotes statistically significant differences in t-tests at p < 0.01 between the mutant and WT.

Figure 7

A working model to illustrate the underlying predicted mechanisms of CsPHYB–CsPIF3/4-regulated hypocotyl elongation. Line thickness is employed as a quantitative indicator to visually represent the intensity of the promoting effect. Thicker lines indicate a stronger intensity of the promoting effect, whereas thinner lines signify a weaker intensity.