Establishment of embryonic shoot-root axis is involved in auxin and cytokinin response during Arabidopsis somatic embryogenesis

Abstract

Auxin and cytokinin signaling participates in regulating a large spectrum of developmental and physiological processes in plants. The shoots and roots of plants have specific and sometimes even contrary responses to these hormones. Recent studies have clearly shown that establishing the spatiotemporal distribution of auxin and cytokinin response signals is central for the control of shoot apical meristem (SAM) induction in cultured tissues. However, little is known about the role of these hormones in root apical meristem (RAM) initiation. Here, we found that the expression patterns of several regulatory genes critical for RAM formation were correlated with the establishment of the embryonic root meristem during somatic embryogenesis in Arabidopsis. Interestingly, the early expression of the WUS-RELATED HOMEOBOX 5 (WOX5) and WUSCHEL genes was induced and was nearly overlapped within the embryonic callus when somatic embryos (SEs) could not be identified morphologically. Their correct expression was essential for RAM and SAM initiation and embryonic shoot-root axis establishment. Furthermore, we analyzed the auxin and cytokinin response during SE initiation. Notably, cytokinin response signals were detected in specific regions that were correlated with induced WOX5 expression and subsequent SE formation. Overexpression of the ARABIDOPSIS RESPONSE REGULATOR genes ARR7 and ARR15 (feedback repressors of cytokinin signaling), disturbed RAM initiation and SE induction. These results provide new information on auxin and cytokinin-regulated apical-basal polarity formation of shoot-root axis during somatic embryogenesis.

Keywords: Arabidopsis; auxin response; cytokinin response; root apical meristem; shoot–root axis; somatic embryogenesis.

FIGURE 1

Expression patterns of WOX5, PLT2, and SCR genes in embryonic calli during somatic embryogenesis. (A–C) Expression patterns of WOX5 indicated by pWOX5::GFP in embryonic calli induced in SEIM for 24 h (A; 83.72%, n = 86), 2 days (B; 84.27%, n = 89) and 3 days (C; 80.21%, n = 96). (D–F) Expression patterns of PLT2 indicated by pPLT2::RFP in embryonic calli induced in somatic embryo-inducing medium (SEIM) for 24 h (D; 87.65%, n = 81), 2 days (E; 84.95%, n = 93) and 3 days (F; 90.53%, n = 95). (G–I) Expression patterns of SCR indicated by pSCR::GFP in embryonic calli induced in SEIM for 24 h (G; 87.36%, n = 87), 2 days (H; 89.58%, n = 96) and 3 days (I; 86.90%, n = 84). CP, cotyledon primordia. Red signals in A–C represent chlorophyll autofluorescence. Scale bars = 80 μm.

FIGURE 2

Relative expression domains of WOX5 and WUS genes. pWOX5::GFP (green) and pWUS::DsRed-N7 (red) signals in embryonic calli induced in SEIM for 24 h (A; 86.81%, n = 91), 2 days (B; 85.39%, n = 89), 3 days (C; 89.66%, n = 87) and 4 days (D; 87.37%, n = 95); CP, cotyledon primordia; Co, cotyledons. Blue signals represent chlorophyll autofluorescence. Scale bars = 80 μm.

FIGURE 3

Functional analysis of both WOX5 and PLT2 during somatic embryogenesis. (A,C,E) Phenotypes of primary somatic embryos (PSE) induction from wild type (WT; A), WOX5 antisense (C) and plt2-1 mutant (E) explants. Arrowheads indicate the PSE. (B,D,F) Phenotypes of SSE induction from WT (B), WOX5 antisense (D) and plt2-1 mutant (F) calli grown on SEIM for 8 days. (G) Expression levels of WOX5 in estradiol-induced 15 days shoots of WT and WOX5 antisense plants. Scale bars = 0.5 mm (A,C,E) and 1.2 mm (B,D,F).

FIGURE 4

Auxin and cytokinin responses in early somatic embryogenesis. (A–D) Auxin response represented by DR5rev:3XVENUS-N7 correlated with WOX5 induction represented by pWOX5::GFP in embryonic calli induced in SEIM for 16 h (A; 88.24%, n = 85), 24 h (B; 83.33%, n = 96), 2 days (C; 87.50%, n = 88) and 3 days (D; 89.66%, n = 87). pWOX5::GFP signals are in green, DR5rev:3XVENUS-N7 fluorescence signals are in red. (E,F) Auxin response represented by DR5rev::GFP correlated with PLT2 induction represented by pPLT2::RFP in embryonic calli induced in SEIM for 3 days (E; 86.73%, n = 98) and 4 days (F; 90.91%, n = 77). DR5rev::GFP fluorescence signals are in green, pPLT2::RFP signals are in red. (G,H) Cytokinin response represented by pARR7::GFP correlated with WUS induction represented by pWUS::DsRed-N7 in embryonic calli induced in SEIM for 2 days (G; 90.43%, n = 94) and 3 days (H; 85.37%, n = 82). pARR7::GFP fluorescence signals are in green, pWUS::DsRed-N7 signals are in red, and chlorophyll autofluorescence is shown in blue. CP, cotyledon primordia; Co, cotyledons. Scale bars = 80 μm.

FIGURE 5

Expression patterns of ARR7 and ARR15 during early somatic embryogenesis. (A–D) Expression patterns of ARR7 indicated by pARR7::GFP in embryonic calli induced in SEIM for 16 h ( A; 93.02%, n = 86), 24 h (B; 90.53%, n = 95), 2 days (C; 85.88%, n = 85) and 3 days (D; 87.63%, n = 97). (E–H) Expression patterns of ARR15 indicated by pARR15::GFP in embryonic calli induced in SEIM for 16 h (E; 88.24%, n = 85), 24 h (F; 82.29%, n = 96), 2 days (G; 94.05%, n = 84) and 3 days (H; 86.96%, n = 92). Red signals represent chlorophyll autofluorescence. Scale bars = 80 μm.

FIGURE 6

Functions of cytokinin signaling on SE induction. (A–D) Phenotypes of SSE induction from 35S::ARR7 (A), 35S::ARR15 (B), ahk2 ahk4 mutant (C), and ahk3 ahk4 mutant (D) calli grown on SEIM for 8 days. (E) WOX5 transcript signals in WT embryonic callus cultured in SEIM for 24 h; 86.15%, n = 35. (F) Dispersed WOX5 transcript signals in ahk3 ahk4 double mutant callus following culture in SEIM for 24 h; 87.30%, n = 33. Scale bars = 1.2 mm (A–D) and 80 μm (E,F).