FRUITFULL Is a Repressor of Apical Hook Opening in Arabidopsis thaliana

Abstract

Plants adjust their architecture to a constantly changing environment, requiring adaptation of differential growth. Despite their importance, molecular switches, which define growth transitions, are largely unknown. Apical hook development in dark grown Arabidopsis thaliana (A. thaliana) seedlings serves as a suitable model for differential growth transition in plants. Here, we show that the phytohormone auxin counteracts the light-induced growth transition during apical hook opening. We, subsequently, identified genes which are inversely regulated by light and auxin. We used in silico analysis of the regulatory elements in this set of genes and subsequently used natural variation in gene expression to uncover correlations between underlying transcription factors and the in silico predicted target genes. This approach uncovered that MADS box transcription factor AGAMOUS-LIKE 8 (AGL8)/FRUITFULL (FUL) modulates apical hook opening. Our data shows that transient FUL expression represses the expression of growth stimulating genes during early phases of apical hook development and therewith guards the transition to growth promotion for apical hook opening. Here, we propose a role for FUL in setting tissue identity, thereby regulating differential growth during apical hook development.

Keywords: AGAMOUS-LIKE8; FRUITFULL; IAA; apical hook opening; light.

Figure 1

Auxin represses light induced growth transition in apical hooks. (A) Kinetics of light triggered apical hook opening in dark grown wild type (WT) seedlings in the maintenance phase (3 DAG) in the presence or absence of 500 nM indole-3-acetic-acid (IAA). (B) Kinetics of light triggered apical hook opening in dark grown pER8:EMPTY and pER8:YUCCA6 seedlings in the presence of 10 µM estradiol. (C) Expression patterns of pGH3.5:GUS in the formation (2 DAG), maintenance (3 DAG), and the opening (4 DAG) phase. (D) Graph depicts mean grey values (mgv) of the pGH3.5:GUS intensity at the inner side of the hook (statistical significance was tested using a 1-way-ANOVA and a Bonferroni post-hoc test, p < 0.05, n ≥ 10 apical hooks per developmental stage. Letters indicate different significance classes). (E,F). Kinetics of light triggered apical hook opening in dark grown WT, gh3.3,5,6 and p35S:GH3.5 seedlings in the maintenance phase (3DAG). (G) DR5 promoter activity as shown by pDR5:GFP signal intensity in 3-day-old (maintenance phase), dark grown WT and p35S:GH3.5 seedlings as visualized using confocal microscopy. Color code (blue to red) depicts (low to high) pDR5:GFP signal intensity. (H) Graph depicts mean grey values of pDR5:GFP intensity at the inner side of the hook. Statistical significance was tested using an unpaired Student’s t-test, *** p < 0.0001, n ≥ 10 seedlings per line.

Figure 2

Putative molecular components in differential growth control in apical hook hooks. (A) Heat map represents genes that are oppositely regulated by the auxin IAA and light in dark grown seedlings. Cut off values were 2-fold expression difference (−1 ≥ log₂ ≥ 1) and p < 0.001. Color code blue to yellow depicts the log₂ values of strongly decreased to strongly increased expression compared to the control condition. (B) Gene regulatory network depicts the transcription factors (orange boxes) which putatively regulate the auxin- and light-regulated candidate genes (green boxes) according to SeqEnrich [16]. Gray lines connect the transcription factors with their putative target genes. The connecting lines with FUL are highlighted in red. (C) Diagram represents the correlation between the expression of FRUITFULL and its target genes in 727 Arabidopsis thaliana natural variations [17] Color code (red to blue) depicts highly negative to highly positive Pearson correlation coefficients. The size of the circles also represents the correlation strength between the expression of FUL and its putative target genes. Non-significant (p ≥ 0.05) correlations are indicated with a cross.

Figure 3

FRUITFULL defines apical hook opening. (A) Expression patterns of pFUL:GUS in the formation (2 DAG), maintenance (3 DAG) and the opening (4 DAG) phase. (B) Graph depicts mean grey values (mgv) of the pFUL:GUS intensity at the inner side of the hook (statistical significance was tested using a 1-way-ANOVA and a Bonferroni post-hoc test, p < 0.05, n ≥ 10 apical hooks per developmental stage. Letters indicate different significance classes). (C–E) Kinetics of light triggered apical hook opening in dark grown WT, ful-7, ful-2, and p35S:FUL seedlings.

Figure 4

FUL-dependent control of SAUR10 in apical hooks. (A) Expression patterns of pSAUR10:GUS in the formation (2 DAG), maintenance (3 DAG) and the opening (4 DAG) phase. (B) Graph depicts mean grey values of the pSAUR10:GUS intensity at the inner side of the hook (statistical significance was tested using a 1-way-ANOVA and a Bonferroni post-hoc test, p < 0.05, n ≥ 10 apical hooks per developmental stage. Letters indicate different significance classes). (C) pSAUR10 promoter activity in 3-day-old (maintenance phase), dark grown WT and ful-7 seedlings. (D) Graph depicts mean grey values of pSAUR10:GUS intensity in the apical hook area. Statistical significance was tested using an unpaired Student’s t-test, *** p < 0.0001, n ≥ 10 seedlings per line. (E) Kinetics of light-triggered apical hook opening in dark grown WT and p35S:SAUR10 seedlings in the early maintenance phase (2-day-old). (F) pGH3.5 promoter activity in 3-day-old (maintenance phase), dark grown WT and ful-7 seedlings. (G) Graph depicts mean grey values of pGH3.5:GUS intensity in the apical hook area. Statistical significance was tested using an unpaired Student’s t-test, *** p < 0.0001, n ≥ 10 seedlings per line. (H) Graph depicts Q-PCR measured relative expression levels of putative FUL target genes in dark grown shoots in the maintenance phase (3 DAG) of ful-7 compared to WT seedlings. The statistical significant difference between WT and ful-7 was demonstrated using a 2-way-ANOVA test including a Bonferroni post-hoc test. p < 0.0001.