Gibberellin biosynthetic deficiency is responsible for maize dominant Dwarf11 (D11) mutant phenotype: physiological and transcriptomic evidence

Abstract

Dwarf stature is introduced to improve lodging resistance and harvest index in crop production. In many crops including maize, mining and application of novel dwarf genes are urgent to overcome genetic bottleneck and vulnerability during breeding improvement. Here we report the characterization and expression profiling analysis of a newly identified maize dwarf mutant Dwarf11 (D11). The D11 displays severely developmental abnormalities and is controlled by a dominant Mendelian factor. The D11 seedlings responds to both GA3 and paclobutrazol (PAC) application, suggesting that dwarf phenotype of D11 is caused by GA biosynthesis instead of GA signaling deficiency. In contrast, two well-characterized maize dominant dwarf plants D8 and D9 are all insensitive to exogenous GA3 stimulation. Additionally, sequence variation of D8 and D9 genes was not identified in the D11 mutant. Microarray and qRT-PCR analysis results demonstrated that transcripts encoding GA biosynthetic and catabolic enzymes ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), and GA 2-oxidase (GA2ox) are up-regulated in D11. Our results lay a foundation for the following D11 gene cloning and functional characterization. Moreover, results presented here may aid in crops molecular improvement and breeding, especially breeding of crops with plant height ideotypes.

Figure 1. Gross morphology of maize D11 mutant.

(A) Phenotype of D11 and wild type (WT). Bar  = 20 cm. (B) Whole plant. To get snapshot of internodes arrangement, leaves and spike were removed manually. Bar  = 20 cm. (C) Plant height. (D) Internodes. Bar  = 5 cm. (E) Internode length. (F) Leaf. Leaves of D11 are slender, dark green, slightly-rolled, and with white margins. Bar  = 5 cm. (G) Roots. Aerial roots of D11 display more sturdy. Bar  = 5 cm. (H) Spike. Spike of D11 degenerates severely. Bar  = 5 cm. (I) Tassel. Bar  = 5 cm. (J) Anther. Anthers of D11 are short and thin. Bar  = 1 mm. (K) Tassel branch number. (L) Length of central axis of tassel. In figures (C), (E), (K), and (L), data are mean ±SD (n = 30). Double asterisks denote significant difference at P≤0.01 level compared with the wild type by Student's t test.

Figure 2. Response of maize D11 mutant to GA₃ and PAC application.

(A) Seedlings of WT and D11 when treated with a 10⁻⁴ M GA₃ solution. Bar  = 10 cm. (B) The second leaf sheath length of WT and D11 (n = 35) when treated with a 10⁻⁴ M GA₃ solution. (C) Seedlings of WT and D11 when treated with a 10⁻⁴ M PAC solution. Bar  = 10 cm. (D) Shoot length of WT and D11 (n = 40) when treated with a 10⁻⁴ M PAC solution. In figures (B) and (D), data are mean ±SD. Double asterisks indicate significant difference at P≤0.01 level compared with untreated samples by Student's t test.

Figure 3. GO clustering of up-regulated DEGs.

(A) GO enrichment analysis according to GO catalogue (GO:0008150 biological process). (B) GO enrichment analysis according to GO catalogue (GO:0005575 cellular component).

Figure 4. DEGs involved in GA biosynthesis and catabolism.

(A) GA biosynthesis and catabolism pathways were briefly diagramed. Transcripts encoding maize GA biosynthetic and catabolic enzymes ZmKAO, ZmGA20ox1, and ZmGA2ox8 are up-regulated in D11. (B) Semi-qRT-PCR validation of elevated transcripts ZmKAO and ZmGA20ox1. The 18S rRNA gene was used as an internal control.