A novel class of gibberellin 2-oxidases control semidwarfism, tillering, and root development in rice

Abstract

Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2beta-hydroxylation: a larger class of C(19) GA2oxs and a smaller class of C(20) GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C(20) GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C(20) GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C(20) GA2oxs were found to cause less severe GA-defective phenotypes than C(19) GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C(20) GA2oxs.

Figure 1.

Schematic Diagram of GA Metabolism and Response Pathways. Conversion of GA₁₂ and GA₅₃ to GA₁₁₀ and GA₉₇, respectively, by 2β-hydroxylation was demonstrated experimentally only for GA2ox6 in this study. GA2ox5, GA2ox6, and GA2ox9 are proposed to have similar functions due to the presence of three conserved motifs unique to C₂₀ GA2oxs. Inactivation of C₁₉-GA precursors, GA₁ and GA₄ by the C₁₉ GA2oxs, GA2ox1 and GA2ox3, in rice was demonstrated experimentally (Sakamoto et al., 2001; Sakai et al., 2003). Bioactive GA positively regulates germination, stem and root elongation, and flower development but negatively regulates OSH1 and TB1 that control tillering. GA also negatively regulates adventitious root development.

Figure 2.

Phylogenetic Tree Based on the Comparison of Plant GA2oxs. Amino acid sequences of 29 GA2oxs from nine plant species (see Supplemental Table 3 online). Plant species: At, Arabidopsis thaliana; Cm, Cucurbita maxima; Ls, Lactuca sativa; Nt, Nicotiana sylvestris; Pc, Phaseolus coccineus; PaPt, Populus alba × P. tremuloides; Ps, Pisum sativum; So, Spinacia oleracea. The scale value of 0.1 indicates 0.1 amino acid substitutions per site.

Figure 3.

Differential Expression of Two Groups of GA2oxs Regulates Flower and Tiller Development. (A) Developmental phases during the life cycle of rice. The timeline is measured in days after imbibition (DAI). (B) Temporal expression patterns of GA2oxs in rice. The last fully expanded leaves were collected from rice plants at different developmental stages. Total RNA was isolated and analyzed by RT-PCR using GA2ox and GA3ox2 gene-specific primers (see Supplemental Table 4 online). The 18S rRNA gene (rRNA) was used as a control. (C) Tiller development during the life cycle of rice. A total of eight plants were used for counting tiller number, and error bars indicate the se of the mean at each time point.

Figure 4.

C₂₀ GA2oxs Could Be Responsible for Regulating Seed Germination. (A) Germination rate of rice seeds reached 100% at 2 DAI. (B) Expression patterns of GA2oxs in rice seedlings between 0 and ∼5 DAI. Total RNA was isolated from embryos at each time point and analyzed by RT-PCR. The 18S rRNA gene (rRNA) was used as a control.

Figure 5.

Severely Dwarfed and Semidwarfed Rice Mutants Obtained by T-DNA Activation Tagging. (A) The severe dwarf mutant GA2ox3_ACT (M77777). (B) The semidwarf mutant GA2ox5Δ335-341_ACT (M27337). (C) The severe dwarf mutant GA2ox6_ACT (M47191). (D) The semidwarf mutant GA2ox9_ACT (M58817). Accumulation of mRNA was analyzed by RT-PCR, with the 18S rRNA gene (rRNA) as a control. T/T and T/W, homozygous and heterozygous mutant, respectively. In the diagram, an asterisk indicates translation start codon, filled box indicates exon, triangle indicates T-DNA, arrowheads indicate position of primers used for RT-PCR analysis, and scale bar represents DNA length for each gene. The box in the triangle indicates the position of the CaMV35S enhancers (next to the left border of T-DNA).

Figure 6.

Overexpression of GA2oxs Has Different Effects on Rice Seed Germination and Seedling Growth. (A) Morphology of T1 seedlings at 18 DAI. (B) Seedling heights of GA2ox5Δ335-341_ACT and GA2ox9_ACT mutants were slightly shorter, while seedlings of GA2ox6_ACT were much shorter than the wild type. Heights of eight plants in each line were measured, and error bars indicate the se of the mean at each time point. (C) Germination rate was normal for the GA2ox9_ACT mutant, slightly delayed for the GA2ox5Δ335-341_ACT mutant, and significantly delayed for the GA2ox6_ACT mutant compared with the wild type. Numbers of seeds for determining germination rates were 154, 50, 156, and 49 for the wild type, GA2ox5Δ335-341_ACT, GA2ox6_ACT, and GA2ox9_ACT, respectively.

Figure 7.

Overexpression of GA2ox5 in Transgenic Rice and Tobacco Causes More Severe Dwarfism Than Overexpression of GA2ox6. (A) and (B) Rice transformed with Ubi:GA2ox5 and Ubi:GA2ox6. (C) to (F) Tobacco transformed with Ubi:GA2ox5 and Ubi:GA2ox6. Transgenic plants showed different degrees of dwarfism compared with the control rice or tobacco transformed with vector only (MS). Photographs of transgenic tobacco were taken at the heading stage ([C] and [D]) and 18 d ([E] and [F]) after sowing of seeds.

Figure 8.

Only Shoot, but Not Root, Growth Is Inhibited by GA Deficiency. (A) Treatment with GA₃ (5 μM) promoted germination and seedling growth of the GA2ox6_ACT mutant (photo taken at 6 DAI). (B) Overexpression of GA2ox6 in rice mutants reduces shoot, but not root, growth. Treatment with GA₃ (5 μM) recovered plant height of the GA2ox6_ACT mutant and root growth of both the wild type and mutant. A total of eight plants at 18 DAI were used for measuring plant height and root length, and error bars indicate the se of the mean. (C) Accumulation of GA2ox6 mRNA in leaves and roots of wild-type and mutant seedlings (at 18 DAI) was not altered by GA₃ treatment. The 18S rRNA gene (rRNA) was used as a control. + and −, presence and absence, respectively.

Figure 9.

GA Deficiency Promotes Early Tillering and Adventitious Root Growth. (A) Swelling on the embryo surface adjacent to the base of the main stem (MS) (positions indicated by arrows) was observed in the GA2ox6_ACT mutant and Ubi:GA2ox5 and Ubi:GA2ox6 transgenic rice (panels 2 to 4) but not in the wild type (panel 1) (photos taken at 3 DAI). (B) First tiller (1T) grew out from the swollen embryo surface of mutant (photo taken at 9 DAI). (C) First and second tillers (1T and 2T) formed in some seedlings of mutant (photo taken at 15 DAI). (D) Each tiller grew out of its own coleoptile, and all new tillers in the mutant had their own adventitious roots (photo taken at 21 DAI). Panel 2 is a higher magnification of the boxed area in panel 1 that reveals coleoptiles (1C and 2C, respectively) and adventitious roots (1R and 2R, respectively) of the main stem and first tiller. (E) Dwarfism and early tillering of seedlings of mutant and transgenic rice compared with the wild type (photo taken at 12 DAI). Panel 2 is a higher magnification of the boxed area in panel 1 that reveals main stem and first tiller.

Figure 10.

GA Deficiency Increases Rice Tiller and Root Numbers. Mutant (GA2ox6_ACT) or transgenic (Ubi:GA2ox5 and Ubi: GA2ox6) seeds germinated on Murashige and Skoog agar medium for 18 DAI. Plant height and tiller and root numbers of 10 plants in each line were determined, and error bars indicate the se.

Figure 11.

Motif III Is Necessary for Activity of GA2ox5 and GA2ox6. (A) Design of constructs encoding the full-length and motif III–truncated GA2ox5 and GA2ox6. Boxes indicate positions of three highly conserved amino acid motifs. The last amino acid residue was shown at the C terminus of deduced polypeptides. (B) Comparison of morphology among transgenic rice overexpressing full-length and motif III–truncated GA2ox5 and GA2ox6 and vector pCAMBIA1301 only (CK).

Figure 12.

GA₃ Suppresses OSH1 and TB1 Expression and Inhibits Tiller and Root Development. (A) Wild-type and GA2ox6_ACT and GA2ox5Δ335-341_ACT mutant seeds were germinated in Murashige and Skoog agar medium with (+) or without (−) 5 μM GA₃. Total RNA was isolated from embryos containing tiller buds at 12 DAI and analyzed by quantitative RT-PCR analysis using primers that specifically amplified rice OSH1 and TB1 cDNAs. RNA levels were quantified and normalized to the level of rRNA. The highest mRNA level was assigned a value of 100, and mRNA levels of other samples were calculated relative to this value. Error bars indicate the se for three replicate experiments. (B) Seedlings used in (A) were photographed prior to RNA isolation. Panels 1 and 2 are higher magnifications of boxed areas for GA2ox5Δ335-341_ACT and GA2ox6_ACT mutants without GA₃ treatment to reveal the main stem (MS) and first tiller (1T).