Spatially selective hormonal control of RAP2.6L and ANAC071 transcription factors involved in tissue reunion in Arabidopsis

Abstract

When grafting or wounding disconnects stem tissues, new tissues are generated to restore the lost connection. In this study, the molecular mechanism of such healing was elucidated in injured stems of Arabidopsis. Soon after the inflorescence stems were incised, the pith cells started to divide. This process was strongly inhibited by the elimination of cauline leaves, shoot apices, or lateral buds that reduced the indole-3-acetic acid supply. Microarray and quantitative RT-PCR analyses revealed that genes related to cell division, phytohormones, and transcription factors were expressed because of incision. Among them, two plant-specific transcription factor genes, ANAC071 and RAP2.6L, were abundantly expressed. ANAC071 was expressed at 1-3 d after cutting exclusively in the upper region of the cut gap, with concomitant accumulation of indole-3-acetic acid. In contrast, RAP2.6L was expressed at 1 d after cutting exclusively in the lower region, with concomitant deprivation of indole-3-acetic acid. The expression of ANAC071 and RAP2.6L were also promoted by ethylene and jasmonic acid, respectively. In transformants suppressing the function of RAP2.6L or ANAC071, the division of pith cells was inhibited. Furthermore, the ethylene signaling-defective ein2 mutant showed incomplete healing. Hence, plant-specific transcription factors differentially expressed around the cut position were essential for tissue reunion of Arabidopsis wounded flowering stems and were under opposite control by polar-transported auxin, with modification by the ethylene and jasmonic acid wound-inducible hormones.

Fig. 1.

Process of tissue reunion in the wounded flowering stem of Arabidopsis. (A–D) Light micrographs of the tissue-reunion process. Arrowheads indicate position of the cut. pi, pith; co, cortex; vb, vascular bundle. (E) Expression of Cyclin B1;1. Each value is the mean ± SE (n = 3). Bars show comparative expression between upper (U; red bar) or lower regions (L; blue bar) of cut stem. The noncut (N; gray bar) control was arbitrarily set to 1, and the mean ± SE is shown (n = 4). (F–I) pCyclin B::GUS plants. (J–M) DR5::GUS plants. Photographs were taken at 1 d (A, F, and J), 3 d (B, G, and K), 5 d (C, H, and L), and 7 d (D, I, and M) after cutting. (Scale bars, 100 μm in A–D, 1 mm in F–M.)

Fig. 2.

Roles of auxin on tissue reunion in Arabidopsis wounded stems. (A and B) Control wild type without decapitation. (C) pin1-1. (D and E) Decapitated wild type. Lanolin paste containing distilled water (D.W.) (A and D), 10⁻³ M TIBA (B), or 10⁻³ M IAA (E) was applied. Arrowheads indicate position of cut. pi, pith; co, cortex; vb, vascular bundle. Images were taken 7 d after stem cutting. (Scale bars in A–E, 100 μm.) (F) Quantification of endogenous IAA in upper (U; red bar), lower (L; blue bar), or noncut region (N; gray bar). Mean ± SE is shown (n = 3).

Fig. 3.

Gene expression of ANAC071 and RAP2.6L TFs and phenotypes of gene-suppressing transformants. (A and B) Gene expression of ANAC071 (A) and RAP2.6L (B). For time-course analysis, the mean ± SE is shown (n = 3). Bars show comparative expression between upper (U; red bar) or lower region (L; blue bar) of wounded stem. The noncut (N; gray bar) control was arbitrarily set to 1, and mean ± SE is shown (n = 4). (C and D) Effects of decapitation and IAA application on the expression of ANAC071 (C) or RAP2.6L (D). Mean ± SE is shown (n = 3). *P < 0.05 (Fisher's test). (E–G) Representative phenotype of SRDX transgenic plants. (E) Wild type. (F) ANAC071-SRDX. (G) RAP2.6L-SRDX. Images were taken 7 d after cutting. Arrowheads indicate position of cut. pi, pith; co, cortex; vb, vascular bundle. (Scale bars in E–G, 100 μm.)

Fig. 4.

Involvement of ethylene in tissue reunion and expression of RAP2.6L and ANAC071. (A) Expression of ACS2. For time-course analysis, the mean ± SE is shown (n = 3). Bars show comparative expression between upper (U; red bar) and lower region (L; blue bar) of cut stem. The noncut (N; gray bar) control was arbitrarily set to 1, and the mean ± SE is shown (n = 4). (B) Light micrograph of ein2 cut stem. Images were taken 7 d after cutting. Arrowheads indicate position of cut. pi, pith; co, cortex; vb, vascular bundle. (Scale bar, 100 μm.) The image is a composite to form the complete figure. (C and D) Relative expression of ANAC071 (C) and RAP2.6L (D) in cut stem (white bar) or noncut stem (gray bar) of wild type, and cut stem (blue bar) or noncut stem (black bar) of ein2. Mean ± SD (n = 2) is shown.

Fig. 5.

Regulation of RAP2.6L expression by JA. (A) Gene expression of LOX2. For time-course analysis, the mean ± SE is shown (n = 3). Bars show comparative expression between upper (U; red bar) and lower region (L: blue bar) of cut stem. The noncut stem control (N; gray bar) was arbitrarily set to 1, and the mean ± SE is shown (n = 4). (B) Effects of methyl jasmonate (MJ) application on the expression of RAP2.6L in the intact flowering stem. The distilled water (D.W.) control was arbitrarily set to 1, and the mean ± SE is shown (n = 3).