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, 8 (1), 36

Down-Regulation of Cytokinin Oxidase 2 Expression Increases Tiller Number and Improves Rice Yield

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Down-Regulation of Cytokinin Oxidase 2 Expression Increases Tiller Number and Improves Rice Yield

Su-Ying Yeh et al. Rice (N Y).

Abstract

Background: Cytokinins are plant-specific hormones that affect plant growth and development. The endogenous level of cytokinins in plant cells is regulated in part by irreversible degradation via cytokinin oxidase/dehydrogenase (CKX). Among the 11 rice CKXs, CKX2 has been implicated in regulation of rice grain yield.

Results: To specifically down-regulate OsCKX2 expression, we have chosen two conserved glycosylation regions of OsCKX2 for designing artificial short hairpin RNA interference genes (shRNA-CX3 and -CX5, representing the 5' and 3' glycosylation region sequences, respectively) for transformation by the Agrobacterium-mediated method. For each construct, 5 independent transgenic lines were obtained for detailed analysis. Southern blot analysis confirmed the integration of the shRNA genes into the rice genome, and quantitative real time RT-PCR and northern blot analyses showed reduced OsCKX2 expression in the young stem of transgenic rice at varying degrees. However, the expression of other rice CKX genes, such as CKX1 and CKX3, in these transgenic lines was not altered. Transgenic rice plants grown in the greenhouse were greener and more vigorous with delayed senescence, compared to the wild type. In field experiments, both CX3 and CX5 transgenic rice plants produced more tillers (27-81 %) and grains (24-67 %) per plant and had a heavier 1000 grain weight (5-15 %) than the wild type. The increases in grain yield were highly correlated with increased tiller numbers. Consistently, insertional activation of OsCKX2 led to increased expression of CKX2 and reduced tiller number and growth in a gene-dosage dependant manner.

Conclusions: Taken together, these results demonstrate that specific suppression of OsCKX2 expression through shRNA-mediated gene silencing leads to enhanced growth and productivity in rice by increasing tiller number and grain weight.

Keywords: Cytokinin oxidase/dehydrogenase; Growth; Productivity; Rice; shRNA.

Figures

Fig. 1
Fig. 1
Molecular analysis of selected primary OsCKX2 transformants (T0) and wild-type (WT) plants. a Detection of transgenes in selected primary transformants. The presence of CX3 (or CX5), hptII and GUS from selected primary transformants was detected by PCR. M: DNA markers, P: positive control (plasmid DNA as template), WT: untransformed wild type, N: negative control (H2O as template). b Expression of OsCKX2 in selected primary transformants and wild type (WT), as assayed by RT-PCR using specific primers. Expression of actin was used as a cDNA loading control
Fig. 2
Fig. 2
Southern blot analysis of hptII, CX3 and CX5 in selected T0 primary rice transformants. The T0 primary transformants selected included two independent transgenic lines (CX3-3 and CX3-8) from transformation with shRNA-CX3 (a, b) and three independent transgenic lines (CX5-1, CX5-3 and CX5-5) from transformation with shRNA-CX5 (c, d). WT: untransformed wild type. Genomic DNA was digested with SacI (a, b) or HindIII (c, d) and hybridized with a 32P-labelled probe corresponding to hptII (a, c), CX3 (b), or CX5 (d). The number of reactive bands in each lane represents the transgene copies in each transgenic line
Fig. 3
Fig. 3
Relative OsCKX2 expression levels in selected T1 CX3- and CX5-suppression lines by qRT-PCR. Total RNA was isolated from young stems of wild type (WT) and selected T1 CX3-suppression lines (2–1–1, 2–1–10, 3–5–2, 3–5–7, 8–2–1, 8–2–3) (a) and CX5-suppression lines (1–2–2, 1–2–4, 1–2–5, 3–6–2, 3–6–6, 3–6–7, 5–2–2, 5–2–3) (b). 17S rRNA was used as an internal control for normalization. The expression level of line 2–1–1 and line 3–6–7 were used as a reference in the comparison of CX3- and CX5-suppression lines, respectively. Values presented were mean +/− SD of 3 replicates of cDNA samples
Fig. 4
Fig. 4
Growth phenotypes of representative T3 CX3- and CX5-Suppression transgenic rice lines grown in the greenhouse. a Mature plants of 4-month-old untransformed wild type (WT) and selected transgenic lines (lines 3 and 8 from transformation with pshRNA-CX3; lines 1, 3 and 5 from transformation with pshRNA-CX5). b Tiller number/plant and plant height analyzed upon harvest. The % data presented are expressed relative to the control (100 %). Values are mean ± SD (n = 7), P <0.01. WT: untransformed wild-type plants; CX3 suppression lines (CX3-3 and CX3-8); CX5 suppression lines (CX5-1, CX5-3 and CX5-5)
Fig. 5
Fig. 5
Agronomic traits of wild-type (WT) and T3 OsCKX2 suppression lines grown in the field. a Panicle number/plant, (b) total grain weight/plant, (c) total grain number/plant and (d) 1000 grain weight. Abbreviations for transgenic lines are the same as described in Fig. 5. Values are mean ± SD (eight replicates with 5 plants/replicate); different letters denoted significance level at P <0.01. The % data presented are expressed relative to the control (100 %)
Fig. 6
Fig. 6
Relative expression levels of OsCKX2 (a) and growth traits (b, c, d) of representative null, heterozygous and homozygous T1 plants derived from OsCKX2-overexpression line, as analyzed by qRT-PCR. a Total RNA was isolated from the leaves of wild type (WT), null lines (1–4, 1–18), CKX2-overexpression heterozygous (1–28, 1–41) and homozygous (1–6, 1–9) lines. 17S rRNA was used as an internal control for normalization. The expression level of WT was used as a reference. Values presented were mean +/− SD of 3 replicates of cDNA samples. b Growth phenotypes of WT, null lines, CKX2-overexpression heterozygous and homozygous lines. (a) Two-month-old plants. b Three-month-old plants. Agronomic traits analyzed upon harvest were (c) panicle number/plant and (d) total biomass/plant
Fig. 7
Fig. 7
Construct of pC1301-CX3 or -CX5 containing a shRNA stem loop for rice transformation. a Diagram of a short hairpin RNA (shRNA) cassette. This cassette comprises inverted repeat sequences (CX3 or CX5) against the target gene (OsCKX2), a spacer fragment (loop) and RNA polymerase III termination sites. b Schematic representation of the transformation constructs (not drawn to scale). shRNA stem loop: 21 nt sense and 21 nt antisense sequences linked by an unpaired loop sequence; Ubiquitin promoter: maize ubiquitin promoter; 35S: CaMV 35S promoter; nos: nopaline synthase terminator; GUS: β-glucuronidase gene; hptII: hygromycin phosphotransferase II gene, LB: left border; RB: right border. c Diagram of self-complementary hairpin siRNA

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