Rice zinc finger protein DST enhances grain production through controlling Gn1a/OsCKX2 expression

Abstract

The phytohormone cytokinin (CK) positively regulates the activity and function of the shoot apical meristem (SAM), which is a major parameter determining seed production. The rice (Oryza sativa L.) Gn1a/OsCKX2 (Grain number 1a/Cytokinin oxidase 2) gene, which encodes a cytokinin oxidase, has been identified as a major quantitative trait locus contributing to grain number improvement in rice breeding practice. However, the molecular mechanism of how the expression of OsCKX2 is regulated in planta remains elusive. Here, we report that the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST) directly regulates OsCKX2 expression in the reproductive meristem. DST-directed expression of OsCKX2 regulates CK accumulation in the SAM and, therefore, controls the number of the reproductive organs. We identify that DST(reg1), a semidominant allele of the DST gene, perturbs DST-directed regulation of OsCKX2 expression and elevates CK levels in the reproductive SAM, leading to increased meristem activity, enhanced panicle branching, and a consequent increase of grain number. Importantly, the DST(reg1) allele provides an approach to pyramid the Gn1a-dependent and Gn1a-independent effects on grain production. Our study reveals that, as a unique regulator of reproductive meristem activity, DST may be explored to facilitate the genetic enhancement of grain production in rice and other small grain cereals.

Fig. 1.

Morphological comparison between wild-type and the reg1 mutant. (A) Gross morphologies of ZF802 and reg1. (B) The panicle morphologies of ZF802 and reg1. (C) Longitudinal sections of the inflorescence meristem of ZF802 and reg1. (Scale bars: A, 20 cm; B, 5 cm; C, 50 μm.) (D) Comparison of grain number per main panicle between ZF802 and reg1. (E) Comparison of inflorescence meristem width between ZF802 and reg1. (F) Comparison of primary branch number per main panicle between ZF802 and reg1. (G) Comparison of secondary branch number per main panicle between ZF802 and reg1. Samples in C and E are inflorescence meristem just after the phase change from vegetative to reproductive stage. Values in D–G are means with SD (n = 30 plants). The double asterisks represent significant difference determined by the Student t test at P < 0.01.

Fig. 2.

Map-based cloning of REG1. (A) The REG1 locus was mapped to an interval between the molecular markers RM6970 and M1 on chromosome 3. (B) Positional cloning narrowed the REG1 locus to a 19-kb region between M3 and M4 at BAC AC133340. Only one gene is predicted in this region by the Rice Annotation Project Database. Numbers on the map indicate the number of recombinants. (C) The REG1 structure and the mutation site in reg1. Predicted ORF and sequence differences between ZF802 and reg1 at the REG1 candidate region are shown. (D) Sequence alignment of the REG1 protein from ZF802 and the reg1 mutant. Identical residues are indicated by dark gray boxes. The EAR motifs in the N-terminal and C-terminal are indicated by purple and pink frames, and the zinc finger domain is indicated by a green frame.

Fig. 3.

The function of reg1 mutation in the inflorescence meristem. (A–E) DST expression during panicle development revealed by RNA in situ hybridization. A is a negative control preparation made with a sense DST probe. B is just after the phase change from vegetative to reproductive stage. C is at the stage of primary branch meristem differentiation. D is at the stage of formation of primary branch primordia. E is at the stage of initiation of spikelet primordia. Arrows in C–E indicate primary branch primordia, secondary branch primodia, and floret primodia, respectively. (F–I) Expression of OsRR1 in inflorescence meristems of ZF802 (F and G) and reg1 (H and I) revealed by RNA in situ hybridization. Samples are inflorescence meristems just after the phase change from vegetative to reproductive stage. F and H are hybridization of OsRR1. G and I are hybridization of sense probes. (J–Q) Expression of OsCKX2 in developing panicles of ZF802 (J–M) and reg1 (N–Q) revealed by RNA in situ hybridization. J and N are just after the phase change from vegetative to reproductive stage. K and O are at the stage of primary branch meristem differentiation. L and P are at the stage of formation of primary branch primordia. M and Q are hybridization of sense probes. (Scale bars: 50 μm). (R) Transcript levels of OsCKX2 in the inflorescence meristems of ZF802 and reg1 revealed by qRT-PCR. Transcript levels of OsCKX2 in ZF802 were arbitrarily set to 1. (S) Transcript levels of OsCKXs in the inflorescence meristems of ZF802 and reg1 revealed by qRT-PCR. Transcript levels of OsCKX8 in ZF802 were arbitrarily set to 1. (T) Comparison of CK levels in the inflorescence meristems of ZF802 and reg1. iP, isopentenyladenine; iPR, iP riboside; iP9G, iP 9-glucoside; Z, zeatin; ZR, zeatin riboside; Z9G, zeatin 9-glucoside. Values are means with SD (n = 3 measurements) in R–T. The asterisks in R–T represent significance difference determined by the Student t test at P < 0.01.

Fig. 4.

DST^REG1 promotes the expression of OsCKX2. (A) Transactivation activity assay in the yeast. BD, GAL4-DNA binding domain; DST^REG1, DST from ZF802; DST^reg1, DST from reg1. Purple box indicates N-terminal EAR motif; green box indicates zinc finger domain; and pink box indicates C-terminal EAR motif. (B) Schematic diagram of the promoter region of OsCKX2. Black box represents DST binding site. Numbers above indicate the distance away from the ATG. Region between the two coupled arrow indicates the DNA fragment used for ChIP-PCR. (C) ChIP assay shows the association of DST^REG1 with the promoter of OsCKX2. Immunoprecipitation was performed with or without myc antibody (no Ab). (D) EMSA assay shows the binding of DST^REG1 to the promoter of OsCKX2. (E) EMSA assay shows the binding of pOsCKX2 with DST^reg1 or DST^REG1ΔZF. In D and E, the arrows indicate the up-shifted bands; FP indicates free probe. (F) DST^REG1 promotes OsCKX2 expression in vivo. Tobacco leaves were transformed with pOsCKX2:LUC plus vector control (1), pUBI:DST^REG1 (2), pUBI:DST^reg1 (3), or pUBI:DST^REG1 and pUBI:DST^reg1 (4). (G) Statistics of luciferase activity in F. These experiments were repeated three times with similar results. (H) qRT-PCR analysis of the expression level of DST in F. Values in G and H are means with SD of three replicates.

Fig. 5.

Effect of the DST^reg1 mutation on grain number formation in different alleles of Gn1a/ OsCKX2. (A) Gross morphologies of NIL R498-DST^REG1 and NIL R498-DST^reg1 at the mature stage. (B) Comparison of the panicle morphologies of NIL R498-DST^REG1 and NIL R498-DST^reg1. (C) Gross morphologies of NIL 93-11-DST^REG1 and NIL 93-11-DST^reg1 at the mature stage. (D) Comparison of the panicle morphologies of NIL 93-11-DST^REG1 and NIL 93-11-DST^reg1. (E) Gross morphologies of NIL NP-DST^REG1 and NIL NP-DST^reg1 at the mature stage. (F) Comparison of the panicle morphologies of NIL NP-DST^REG1 and NIL NP-DST^reg1. (Scale bars: A, C, and E, 20 cm; B, D, and F, 5 cm.)