The WUSCHEL-related homeobox gene WOX11 is required to activate shoot-borne crown root development in rice

Abstract

In rice (Oryza sativa), the shoot-borne crown roots are the major root type and are initiated at lower stem nodes as part of normal plant development. However, the regulatory mechanism of crown root development is poorly understood. In this work, we show that a WUSCHEL-related Homeobox (WOX) gene, WOX11, is involved in the activation of crown root emergence and growth. WOX11 was found to be expressed in emerging crown roots and later in cell division regions of the root meristem. The expression could be induced by exogenous auxin or cytokinin. Loss-of-function mutation or downregulation of the gene reduced the number and the growth rate of crown roots, whereas overexpression of the gene induced precocious crown root growth and dramatically increased the root biomass by producing crown roots at the upper stem nodes and the base of florets. The expressions of auxin- and cytokinin-responsive genes were affected in WOX11 overexpression and RNA interference transgenic plants. Further analysis showed that WOX11 directly repressed RR2, a type-A cytokinin-responsive regulator gene that was found to be expressed in crown root primordia. The results suggest that WOX11 may be an integrator of auxin and cytokinin signaling that feeds into RR2 to regulate cell proliferation during crown root development.

Figure 1.

WOX11 Is Expressed in Cell Division Regions in Roots and Shoots. (A) Detection of WOX11 transcript by RT-PCR in callus (Ca), roots (Ro), seedlings (Se), internodes (In), mature leaves (Le), flag leafs (Fl), and panicles (Pa). PCRs without prior reverse transcription (−RT) are included. Actin transcripts were detected as controls. (B) to (I) In situ hybridization detection of WOX11 transcripts in the primary root with an antisense (B) or a sense probe (C), in crown root initials (D), in growing crown primordia ([E], longitudinal section; [F], transverse section), in a mature crown root (G), and in the SAM with an antisense (H) or a sense probe (I). Bars = 25 μm in (B), (C), (G), (H), and (I) and 50 μm in (D) to (F). Arrows indicate emerging crown roots. (J) to (M) GUS activity in a 7-d-old WOX11p-GUS transgenic seedling: a primary root tip (J); lateral roots (K); and a transverse section of the root meristematic zone (L). (M) shows a transverse section through a lateral root. (N) DR5-GUS expression profile in root. Bars = 25 μm in (J), (L), and (N) and 50 μm in (K) and (M). (O) to (S) Detection of WOX11p-GUS expression during the course of crown root initiation. GUS activity was detected in emerging crown roots ([R] and [S]) but not at the early stages during crown root initiation ([O] to [Q]). Arrowheads indicate crown root initials. Bars = 25 μm.

Figure 2.

Characterization of Two T-DNA Insertion Lines of WOX11. (A) Schematic representation of the WOX11 locus. Relative nucleotide positions of the coding region are indicated (with the initiation ATG codon assessed as 1). The T-DNA insertion positions in the second exon are indicated by open arrows. The positions of the primer L2 and R1 (corresponding to the T-DNA left and right borders, respectively) and the forward (F2) and reverse (R2) WOX11 primers are indicated by arrows. Positions of primers (F3 and R3) used to detect WOX11 expression are indicated. Filled boxes, exons; open boxes, untranslated exons; thick lines, introns. (B) Detection of WOX11 transcripts in the root [WT(R)] or seedlings [WT(S)] of the wild type or seedlings of wox11-1 and wox11-2. Two different samples (m1 and m2) of each mutant line were used for the RT-PCR analysis. Actin transcripts were amplified as controls. (C) to (E) Phenotypes of wox11-1 (on the right of each panel) compared with the wild type (left) at 7 d (C) and 14 d ([D] and [E]) after germination. (F) to (H) Phenotype of wox11-2 (on the right of each panel) compared with the wild type (left) at 7 d (F) and 14 d ([G] and [H]) after germination. PR, primary root; CR, crown roots. Bars = 1 cm in (C), (D), (F), and (G), 5 cm in (E), and 2 cm in (H). [See online article for color version of this figure.]

Figure 3.

Analysis of WOX11 RNAi Transgenic Plants. (A) Schematic representation of the WOX11 cDNA. The coding region is boxed. The slash region in the box corresponds to the conserved homeodomain. The cDNA segment used to construct the RNAi vector is indicated. UTR, untranslated region. (B) Real-time RT-PCR analysis of WOX11 transcripts in the wild type and six RNAi transgenic lines. The PCR signals were normalized with actin transcripts. Transcript levels from the wild type were set at 1. Data are means ± sd (n = 3) (C) Comparison of 1-week-old seedlings between the wild type (left) and the RNAi line RW4 (right). (D) Comparison of 2-week-old seedlings between the wild type (left) and the RNAi line RW4 (right). (E) Comparison of mature plants between the wild type (left) and the RNAi line RW4 (right). LR, lateral root; CR, crown root; PR, primary root. Bar = 1 cm in (C) and (D) and 30 cm in (E). [See online article for color version of this figure.]

Figure 4.

Overexpression of WOX11-Induced Ectopic Crown Roots in Transgenic Rice. (A) Detection of WOX11 transcripts by RT-PCR in eight phenotypic transgenic lines (OW). Actin transcripts were detected as controls. (B) Comparison of 1-week-old seedlings of the wild type (left) with the overexpression plant (right). Inset: enlarged view of the transgenic root showing precocious production of lateral roots on the crown roots. (C) Comparison of 2-week-old roots between the wild type (left) and the overexpression plants (right). (D) An overexpression plant producing a large number of crown roots, showing roots growing out from the shoot. (E) Comparison of a wild type (left) and an overexpression plant (right) at the four-leaf stage. (F) Ectopic crown roots produced on the lower nodes of the overexpression plants (indicated by arrows). (G) Ectopic crown roots produced on the upper nodes (indicated by arrows). (H) Ectopic crown roots produced in the panicles. (I) Two ectopic crown roots produced at the base of a floret in an overexpression plant. Pictures were taken from lines OW1, OW2, or OW7, which showed similar phenotypes. LR, lateral root; CR, crown-borne root; PR, primary root. Bars = 2 cm.

Figure 5.

Crown root growth rates in wild-type and WOX11 RNAi, mutant, and over-expression plants. Hematoxylin-stained cross sections of the coleoptilar node of wild-type, WOX11 RNAi (RW4), wox11-1 mutant, and WOX11-overexpressing (OW7) seedlings at 3, 5, 7, or 21 d after germination as indicated. Arrows indicate emerging crown root initials. Bars = 50 μm.

Figure 6.

Kinetics of Induction of WOX11 in Response to Plant Hormones IAA, NAA, and 6-BA. The transcript levels of WOX11 in 10-d-old light-grown wild-type seedlings treated with IAA, NAA, or 6-BA for the indicated times were plotted as the relative expression (fold) of water-treated seedlings during the same durations. The PCR signals were normalized with those of the actin transcripts. Data are means ± sd (n = 3).

Figure 7.

Expression of Auxin- and Cytokinin-Responsive Genes in Wild-Type, WOX11 RNAi, and Overexpression Plants. (A) Relative expression levels determined by real-time RT-PCR of four rice Aux/IAA genes (IAA5, IAA11, IAA23, and IAA31) in three WOX11 overexpression (OW) or three RNAi (RW) lines compared with the wild type. (B) Relative expression levels of six rice type-A RR genes. A nonphenotypic RNAi transgenic sibling (TS) was included for comparison. The PCR signals were normalized with those of the actin transcripts. Transcript levels from the wild type were set at 1. Data are means ± sd of three biological replicates.

Figure 8.

In Situ Hybridization Detection of RR2 Transcripts in Crown Roots. (A) to (C) In coleoptilar node sections of a 5- (A), 7- (B), or 12-d-old (C) seedlings. (D) and (E) In mature crown roots with the antisense (D) or sense (E) probes. (F) In a coleoptilar node section of a 21-d-old wox11-1 seedling. (G) In a coleoptilar node section of a 5-d-old WOX11 overexpression seedling (OW7). Bars = 50 μm.

Figure 9.

WOX11 Interacts with the RR2 Gene in Vitro and in Vivo. (A) Gel shift assays of WOX11 protein binding to a promoter sequence (S1) of RR2 containing the WOX binding site (underlined) or a mutant version of the promoter (S2). E. coli–produced WOX11 protein was incubated with ³²P-labeled S1 or S2 in the absence or presence of 50 or 100 M excess of the corresponding cold probes and analyzed by electrophoresis. The shifted band is indicated by an arrow. (B) ChIP analysis of transgenic plants expressing a WOX11-GFP fusion protein. Nuclei from two WOX11-GFP expression lines (3A and 3B) and the wild-type plants were immunoprecipitated by anti-GFP or without antibody. The precipitated chromatin fragments were analyzed by 26 cycles of PCR using four primer sets (P1 to P4) amplifying four RR2 promoter regions as indicated. The relative nucleotide positions of the putative WOX binding site are indicated (with the initiation ATG codon assessed as +1). One-tenth of the input chromatin was analyzed as controls. (C) ChIP analysis of WOX11-GR transgenic plants using antibodies against mouse glucocorticoid receptor. Nuclei from two WOX11-GR expression lines (WG3 and WG5) and wild-type plants treated with (+) or without (–) DEX were immunoprecipitated by anti-GR or without antibody. The precipitated chromatin fragments were analyzed by 28 cycles of PCR using three primer sets (P1 to P3) as indicated. One-tenth of the input chromatin was analyzed as controls.