A WUSCHEL Homeobox Transcription Factor, OsWOX13, Enhances Drought Tolerance and Triggers Early Flowering in Rice

Abstract

Plants have evolved strategies to cope with drought stress by maximizing physiological capacity and adjusting developmental processes such as flowering time. The WOX13 orthologous group is the most conserved among the clade of WOX homeodomain-containing proteins and is found to function in both drought stress and flower development. In this study, we isolated and characterized OsWOX13 from rice. OsWOX13 was regulated spatially in vegetative organs but temporally in flowers and seeds. Overexpression of OsWOX13 (OsWOX13-ov) in rice under the rab21 promoter resulted in drought resistance and early flowering by 7-10 days. Screening of gene expression profiles in mature leaf and panicles of OsWOX13-ov showed a broad spectrum of effects on biological processes, such as abiotic and biotic stresses, exerting a cross-talk between responses. Protein binding microarray and electrophoretic mobility shift assay analyses supported ATTGATTG as the putative cis-element binding of OsWOX13. OsDREB1A and OsDREB1F, drought stress response transcription factors, contain ATTGATTG motif(s) in their promoters and are preferentially expressed in OsWOX13-ov. In addition, Heading date 3a and OsMADS14, regulators in the flowering pathway and development, were enhanced in OsWOX13-ov. These results suggest that OsWOX13 mediates the stress response and early flowering and, thus, may be a regulator of genes involved in drought escape.

Keywords: Hd3a; OsWOX13; drought; early flowering; escape; vascular tissue.

Fig. 1. Domain analyses of WOX13 OG proteins. The evolutionary history was inferred from homeodomain sequences (Supplementary Fig. S1) using the neighbor-joining method

The evolutionary distances were computed using the Poisson correction method and are expressed as the number of amino acid substitutions per site. Members of WOX1, WOX8 and WOX13 orthologous groups are connected by blue, red and green lines; respectively. Arabidopsis genes in each OG are represented by OG-specific colored characters. Os01g0818400/OsWOX13 is colored and boxed.

Fig. 2. Histochemical analysis of OsWOX13p::GUS reporter gene expression in plant development

(A) Mature and (B) 2-day imbibed seeds. (C) A 5-day seedling. (D, E) Magnificent of a leaf blade (D) and longitudinal cut through a seed and stem (E) of a 5-day seedling. The white arrow in (E) indicates the position where GUS expression stopped on the crown root. (F) Cross-section of a crown root from a 7-day seedling. (G) Lateral root initiation in a 7-day seedling crown root. (H, I) Junctions with developed (H) and developing (I) lateral root in a 7-day seedling radicle. (J) Six-week-old stem with all developed leaves removed. The arrow shows the position of an auxiliary tiller where it is cross cut (K) and 12-μm-sectioned (L). The section in (L) was stained with toluidine blue. (M) The 12-μm cross-section of the SAM sample was stained with toluidine blue. (N) Hand section of a node from an unelongated stem. (O) Hand section of node N^th counted upward along an elongated stem. In node N^th, DVs only surround and connect to enlarged large VBs of leaf N^th at NVA. VBs of leaf (N+1)^th are not enlarged and only pass through the node. (P) Panicles at the very early stage of inflorescent formation. (Q to T) Floret organs at stage (Q) 1 cm, (R) 12 cm, and (S) before and (T) after pollination. (U, left to right) Seeds at 2, 4, 6, 8, and 10DAP. Scale bar = 0.2 cm (a, b, c, e, j, t); 50 μm (F, H) or 200 μm (others). sc: scutellum, pl: primary leaf, ra: radicle, cr: crown root, me: mesocotyl, mv: main vien, NVA: nodal vascular anatomoses, VBs: vascular bundles, P_LN and X_LN: phloem and xylem of enlarged large VBs of leaf N^th, S_N: enlarged small VBs of leaf N^th, L/S_N+1: large/small VBs of leaf (N+1)^th, DVs: diffuse vascular bundle, SAM: shoot apical meristem, IM: inflorescence meristem, DAP: day-after-pollination.

Fig. 3. Induction of OsWOX13 by abiotic stressors

(A) RT-PCR analysis confirmed that Os01g0818400/OsWOX13 were moderately up-regulated under drought stress in leaf and root. (B) Leaves of 14-d greenhouse-grown seedlings were collected directly (NC) or subsequently the whole plant was air-dried (dry) in a growth chamber. Another set was removed from soil to water and kept in a chamber at 28°C (H₂O), 42°C (heat) or 4°C (cold). Then, 400 mM NaCl was also added to the water to induce salt stress (salt). All leaves were collected after 3 h.

Fig. 4. Seedlings of transgenic rice under dehydration and OsWOX13 expression pattern

Seeds were germinated in soil (12 seeds/5x5x6 cm pot). Drought stress was conducted by water-withdrawal for 1 day in 2-week-old seedling followed by rewatering for 2 days (A). Leaves were collected, and the expression of OsWOX13 was examined (B). Bands were quantified and divided to assess the corresponding ubi to obtain the relative ratio to ubiquitin given as the number under each band. Given that ubi is the constitutive primer, all bands of ubi were calibrated to 1.00.

Fig. 5. Overexpression of OsWOX13 (rab21 promoter) showed early flowering

(A) Three T₃lines showed the early flowering phenotype in the field. Wild type Ilmi showed heading around Aug. 17th in the irrigated field, and the transgenic plants showed 7–10-d earlier flowering compared with Ilmi. (B) Normal distribution of heading dates of transgenic and wild type plants. (C) Mature leaves were collected at day 40, 33, 25 (Δ40, Δ33, Δ25; respectively) prior to the heading date. The 1 cm (P1cm, Δ19) and 8 cm (P8cm, Δ12)-long panicles were also included. The transgenic line 8-4-1 was used as a representative (OsWOX13-ov). (D) The expression level of flowering-related genes by qPCR with tubulin as the expression standard.

Fig. 6. Analysis of the putative DNA binding sequence of OsWOX13

(A) LOGO of cis-acting element from the Q9-PBM results. (B) Sequences of non-biotinylated competitors (Cp) used in (c). (C) EMSA-based competition analysis. (D) DNA binding affinity graph of OsWOX13 protein with ATTG-2x non-biotinylated probe in an EMSA-based test.