CYTOKININ OXIDASE/DEHYDROGENASE4 Integrates Cytokinin and Auxin Signaling to Control Rice Crown Root Formation

Abstract

Crown roots constitute the majority of the rice (Oryza sativa) root system and play an important role in rice growth and development. However, the molecular mechanism of crown root formation in rice is not well understood. Here, we characterized a rice dominant mutant, root enhancer1 (ren1-D), which was observed to exhibit a more robust root system, increased crown root number, and reduced plant height. Molecular and genetic analyses revealed that these phenotypes are caused by the activation of a cytokinin oxidase/dehydrogenase (CKX) family gene, OsCKX4. Subcellular localization demonstrated that OsCKX4 is a cytosolic isoform of CKX. OsCKX4 is predominantly expressed in leaf blades and roots. It is the dominant CKX, preferentially expressed in the shoot base where crown root primordia are produced, underlining its role in root initiation. OsCKX4 is induced by exogenous auxin and cytokinin in the roots. Furthermore, one-hybrid assays revealed that OsCKX4 is a direct binding target of both the auxin response factor OsARF25 and the cytokinin response regulators OsRR2 and OsRR3. Overexpression and RNA interference of OsCKX4 confirmed that OsCKX4 plays a positive role in crown root formation. Moreover, expression analysis revealed a significant alteration in the expression of auxin-related genes in the ren1-D mutants, indicating that the OsCKX4 mediates crown root development by integrating the interaction between cytokinin and auxin. Transgenic plants harboring OsCKX4 under the control of the root-specific promoter RCc3 displayed enhanced root development without affecting their shoot parts, suggesting that this strategy could be a powerful tool in rice root engineering.

Figure 1.

Phenotypes of the ren1-D mutant. A, Root systems of wild-type (WT) and ren1-D seedlings at 7 DAG. Bar = 4 cm. B, Cross sections of the lower coleoptilar nodes of wild-type and ren1-D seedlings at 2 DAG. Red arrows indicate crown root primordia. Bars = 100 μm. C, Phenotypes of wild-type and ren1-D seedlings at 4 DAG. Bar = 1 cm. D, Phenotypes of wild-type and ren1-D plants at the heading stage. Bar = 10 cm. E, Number of crown roots of wild-type and ren1-D seedlings at 7 DAG. Data are means ± sd (n > 15 seedlings; **P < 0.01). F, Root lengths of wild-type and ren1-D seedlings at 7 DAG. Data are means ± sd (n > 15 seedlings; *P < 0.05). G, Root dry weight of wild-type and ren1-D seedlings at 7 DAG. Data are means ± sd (n > 15 seedlings; *P < 0.05). H, Shoot lengths of wild-type and ren1-D seedlings at 7 DAG. Data are means ± sd (n > 15 seedlings; **P < 0.01).

Figure 2.

Cytokinin content and response in ren1-D mutants. A, Quantification of endogenous cytokinin content in 2-week-old roots. iP, Isopentenyladenine; tZ, trans-zeatin; DHZ, dihydrozeatin; iPR, isopentenyladenine riboside; tZR, trans-zeatin riboside; DHZR, dihydrozeatin riboside; iP9G, isopentenyladenine 9-glucoside; tZ9G, trans-zeatin 9-glucoside. Data are means ± sd (n = 3). B, Kinetin-treated wild-type (WT) and ren1-D seedlings at 6 DAG cultured in one-half-strength MS medium containing 0, 0.1, 1, or 10 μm kinetin (from left to right). Bar = 3 cm. C, Statistics of kinetin treatment on shoot growth in wild-type and ren1-D seedlings. D, Relative expression levels of rice type A response regulator genes in ren1-D mutants. Ubq2 was used as an internal control. Data are means ± sd (n = 3).

Figure 3.

Expression pattern of OsCKX4 and subcellular localization of OsCKX4. A, Analysis of OsCKX4 expression levels in various tissues by qRT-PCR. Ubq2 was used as an internal control. Data are means ± sd (n = 3). B, OsCKX4 promoter:GUS expression patterns in transgenic rice plants: (1) primary root and lateral root; the red arrow indicates the lateral root; (2) crown root; (3) root tip; (4) shoot base of the young seedling; the red arrow indicates the shoot base; (5) mature floret; (6) leaf sheath; (7) leaf blade; (8) stem. C, Relative transcript levels of OsCKXs at the base of shoots revealed by qRT-PCR. Ubq2 was used as an internal control. Data are means ± sd (n = 3). D, Subcellular localization of 35S:GFP (top row) and 35S:OsCKX4-GFP (bottom row) in rice protoplast cells. DIC indicates the differential interference contrast, and Merged indicates the merger of GFP and DIC images. Bars = 10 μm.

Figure 4.

Auxin and cytokinin regulate OsCKX4 expression in roots. A, Structure of the OsCKX4 gene promoter region. The positions of cytokinin response elements (5′-AGATT-3′) and auxin response elements (5′-TGTCTC-3′) are highlighted. Numbers below each element indicate the distance away from the ATG. B, qRT-PCR analysis of OsCKX4 expression after treatment with exogenous 6-BA (5 μm). The transcript level of OsCKX4 in untreated seedlings was set as 1.0. Ubq2 was used as an internal control. Data are means ± sd (n = 3). C, qRT-PCR analysis of OsCKX4 expression after treatment with exogenous IAA (10 μm). The transcript level of OsCKX4 in untreated seedlings was set as 1.0. Ubq2 was used as an internal control. Data are means ± sd (n = 3). D, GUS staining with 6-BA or IAA treatment in OsCKX4 promoter:GUS rice lines. The roots from the OsCKX4 promoter:GUS transgenic lines were immersed into 6-BA (5 μm) or IAA (10 μm) for 1 h before GUS staining. Bars = 1 cm. E, Yeast one-hybrid assays to test whether the transcription factors OsARF1, OsARF12, OsARF25, ORR1, ORR2, ORR3, and ORR6 bind to the OsCKX4 promoter. Empty vector expressing the activation domain (AD) alone was used as a negative control.

Figure 5.

Analysis of OsCKX4-RNAi transgenic plants. A, qRT-PCR analysis of OsCKX4 transcripts in the wild type (WT) and two independent OsCKX4-RNAi transgenic lines. The transcript level of OsCKX4 in the wild type was set as 1.0. Ubq2 was used as an internal control. Data are means ± sd (n = 3). B, Comparison of 1-week-old seedlings between the wild type and two independent OsCKX4-RNAi lines. Left, The wild type; center, RNAi line 32; right, RNAi line 42. Bar = 2 cm. C, Comparison of the root phenotypes of 2-week-old seedlings between the wild type and two independent OsCKX4-RNAi lines. Left, The wild type; center, RNAi line 32; right, RNAi line 42. Bar = 5 cm.

Figure 6.

Auxin responses in ren1-D mutants. A, Gravisensitivity of seedling roots. Wild type (WT) and ren1-D seedlings were grown vertically for 3 d and then rotated 90°. B, Statistics for the root tip angles in A at 24 h after reorientation. C, Effects of NPA treatment on shoot and root growth in the wild type and ren1-D mutants 6 d after germination and then cultured in MS medium containing 0, 0.5, or 1 μm NPA. Bars = 2 cm. D, Statistics of NPA treatment on root growth in the wild type and ren1-D mutants. E, Measurement of free IAA content in roots of the wild type and ren1-D mutants. **P < 0.01. F, qRT-PCR analyses revealed the expression of auxin biosynthesis genes in the roots of ren1-D mutants. Ubq2 was used as an internal control. Data are means ± sd (n = 3). **P < 0.01. G, qRT-PCR analyses revealed the expression of auxin transporter genes and crown root development-related genes in the roots of ren1-D mutants. Ubq2 was used as an internal control. Data are means ± sd (n = 3). *P < 0.05, **P < 0.01.

Figure 7.

Root-specific expression of OsCKX4. A, Map of the binary vector used for generating RCc3:OsCKX4 transgenic plants. Hpt, Hygromycin phosphotransferase gene, used as a selection marker; LB, left border; OCS, octopine synthase terminator; RB, right border. B, Three-week-old wild-type (WT; left) and RCc3:OsCKX4 (center, RCc3-1; right, RCc3-2) seedlings. Bar = 5 cm. C, Enlarged view of the root phenotypes corresponding to the frame in B. Bar = 2 cm. D, Analysis of OsCKX4 expression levels in roots and shoots of wild-type and RCc3:OsCKX4 plants by qRT-PCR. E, Root dry weight of 3-week-old wild-type and RCc3:OsCKX4 plants. *P < 0.05.

Figure 8.

Working model for OsCKX4 function in rice crown root formation. The OsCKX4 protein catalyzes the irreversible degradation of cytokinins. Reduction of the cytokinin status in planta causes the formation of a larger root system. The OsCKX4 transcriptional activity is regulated by auxin and cytokinin through ORR- and OsARF-mediated signaling pathways. OsHKs, rice His kinases, are genes of a cytokinin receptor family. TSS, Transcription start site.