The regulation of the Z- and G-box containing promoters by light signaling components, SPA1 and MYC2, in Arabidopsis

Abstract

Although many transcription factors and regulatory proteins have been identified and functionally characterized in light signaling pathways, photoperception to transcription remains largely fragmented. The Z-box is one of the LREs (Light responsive elements) that plays important role in the regulation of transcription during light-controlled Arabidopsis seedling development. The involvement of photoreceptors in the modulation of the activity of the Z-box containing promoters has been demonstrated. However, the role of downstream signaling components such as SPA1 and MYC2/ZBF1, which are functionally interrelated, remains unknown. In this study, we have investigated the regulation of the Z-box containing synthetic and native promoters by SPA1 and MYC2 by using stable transgenic lines. Our studies suggest that SPA1 negatively regulates the expression of CAB1 native promoter. MYC2 negatively regulates the activity of Z- and/or G-box containing synthetic as well as native promoters irrespective of light quality. Moreover, MYC2 negatively regulates the expression of Z/G-NOS101-GUS even in the darkness. Furthermore, analyses of tissue specific expression in adult plants suggest that MYC2 strongly regulates the activity of Z- and G-box containing promoters specifically in leaves and stems. In roots, whereas MYC2 positively regulates the activity of the Z-box containing synthetic promoter, it does not seem to control the activity of the G-box containing promoters. Taken together, these results provide insights into SPA1- and MYC2-mediated transcriptional regulation of the Z- and G-box containing promoters in light signaling pathways.

Figure 1. Effect of spa1 mutation on the regulation of Z/NOS101–GUS and CAB1-GUS promoters under different wavelengths of light.

(A), The consensus DNA sequences of LREs (Z, GATA, GT1 and G-box) derived from different light responsive promoters. (B–C), In each panel, wild-type (WT) and spa1 mutant seedlings carrying respective transgene were shown on the left and right, respectively. GUS staining patterns of 6-day-old wild-type and spa1 mutant seedlings carrying Z/NOS101-GUS (B) and CAB1-GUS (C) transgene grown in different light (white light (WL), far-red light (FR), red light (RL), and blue light (BL) or dark (D)) conditions as indicated. (D), GUS activities of six-day-old constant D, WL, RL, FR and BL grown seedlings carrying Z/NOS101-GUS transgene in wild-type and spa1 mutant backgrounds. Error bars represents SD (n = 3). ** P≤0.01 for values significantly differ from corresponding light conditions in wild-type. (E), GUS activities of six-day-old constant D, WL, RL, FR and BL grown seedlings carrying CAB1-GUS transgene in wild-type and spa1 mutant backgrounds. Error bars represents SD (n = 3). *** P≤0.001 for values significantly differ from corresponding light conditions in wild-type. All the above experiments were performed at least thrice with similar results.

Figure 2. Effect of atmyc2/zbf1 mutation on the regulation of Z-box containing promoters.

(A–B), In each panel, wild-type and atmyc2/zbf1 mutant seedlings carrying respective transgene were shown on the left and right, respectively. GUS staining patterns of six-day-old wild-type and atmyc2 seedlings carrying Z/NOS101-GUS (A) and CAB1-GUS (B) transgene grown in different light or dark conditions as indicated. (C–D), GUS activities of wild-type and atmyc2 seedlings carrying Z/NOS101-GUS (C) and CAB1-GUS (D) transgene grown in different light or dark conditions as indicated. Error bars represents SD (n = 3). ** P≤0.01 and *** P≤0.001 for values significantly differ from WT in respective growth conditions. All the above experiments were performed at least thrice with similar results.

Figure 3. Light-mediated induction of CAB1-GUS in atmyc2 mutatants.

(A), Four-day-old dark-grown seedlings carrying CAB1-GUS transgene were exposed to WL for 0, 6, 12 and 24 h and GUS activities were measured. Error bars represents SD (n = 3). (B), Four-day-old WL grown seedlings carrying CAB1-GUS transgene were exposed to dark for 0, 6, 12 and 24 h and GUS activities were measured. Error bars represents SD (n = 3). All the above experiments were performed at least thrice with similar results.

Figure 4. Effect of atmyc2/zbf1 mutation on the regulation of G-box containing promoters.

(A–B), In each panel, wild-type and atmyc2/zbf1 mutant seedlings carrying respective transgene were shown on the left and right, respectively. GUS staining patterns of six-day-old wild-type and atmyc2 seedlings carrying G/NOS101-GUS (A) and G-GATA/NOS101-GUS (B) transgene grown in different light or dark conditions as indicated. (C–D) GUS activities of wild-type and atmyc2 seedlings carrying G/NOS101-GUS (C) and G-GATA/NOS101-GUS (D) transgene grown in different light or dark conditions as indicated. Error bars represents SD (n = 3). ** P≤0.01 and *** P≤0.001 for values significantly differ from WT in respective growth conditions. All the above experiments were performed at least thrice with similar results.

Figure 5. Effect of atmyc2/zbf1 mutation on the tissue specific expression of Z- box containing promoters in adult plants.

In each panel (A-H) wild-type and atmyc2 seedlings carrying respective transgene are shown on the left and right, respectively. For tissue specific staining, 35-days-old adult plants grown in 14 h Light/10 h Dark cycle were used for the experiment. (A–D) The GUS staining patterns of Z/NOS-GUS transgene from leaf (A), stem (B), flower (C) and root (D). (E–H) The GUS staining patterns of CAB1-GUS transgene from leaf (E), stem (F), flower (G), and root (H). (I–J) GUS activities of 35-day-old wild-type and atmyc2 plants carrying Z/NOS101-GUS (I) and CAB1-GUS (J) transgene. Error bars represents SD (n = 3). Error bars represents SD (n = 3). ** P≤0.01 and *** P≤0.001 for values significantly differ from WT in respective tissues. All the above experiments were performed at least thrice with similar results.

Figure 6. Effect of atmyc2/zbf1 mutation on the tissue specific expression of G- box containing promoters in adult plants.

(A–D)The GUS staining patterns of G/NOS101-GUS transgene from leaves (A), stem (B),flower (C), and root (D). (E–H)The GUS staining patterns of G-GATA/NOS101-GUS transgene from leaves (E), stem (F), flower (G), and root (H). (I–J) Comparison of GUS activities of 35-day-old wild-type and atmyc2 seedlings carrying G/NOS-GUS (I) and G-GATA/NOS101-GUS (J) transgene. Error bars represents SD (n = 3). Error bars represents SD (n = 3). ** P≤0.01 for values significantly differ from WT in respective in respective tissues. All the above experiments were performed at least thrice with similar results.

Figure 7. Mode of regulation of Z- and/or G-box containing promoters by MYC2 and SPA1.

MYC2 inhibits the expression of SPA1 . MYC2 negatively regulates the Z- and/or G-box containing promoters irrespective of light quality by directly binding to the promoters. Whereas SPA1 positively regulates the Z-box containing promoter, it negatively regulates the activity of native CAB1 in a wavelength independent manner through unknown regulatory protein (X) during photomorphogenesis.