Gibberellins control fruit patterning in Arabidopsis thaliana

Abstract

The Arabidopsis basic helix-loop-helix (bHLH) proteins INDEHISCENT (IND) and ALCATRAZ (ALC) specify tissues required for fruit opening that have major roles in seed dispersal and plant domestication. Here, we show that synthesis of the phytohormone gibberellin is a direct and necessary target of IND, and that ALC interacts directly with DELLA repressors, which antagonize ALC function but are destabilized by gibberellin. Thus, the gibberellin/DELLA pathway has a key role in patterning the Arabidopsis fruit, and the interaction between DELLA and bHLH proteins, previously shown to connect gibberellin and light responses, is a versatile regulatory module also used in tissue patterning.

Figure 1.

The gibberellin biosynthetic gene GA3ox1 is a direct target of IND. (A) Mature wild-type Arabidopsis fruit and schematic cross-section showing the different tissues. The regulatory network controlling the development of fruit tissues is also indicated. Expression of the valve margin identity genes (IND, ALC, and SHP1/2) is restricted to the valve margins by the FUL and RPL activities in the valve and the replum, respectively. Bar, 1 mm. (B) Transcript profiling assay using 7-d-old 35S∷IND:GR seedlings before (−DEX) and after (+DEX) 6 h of DEX treatment, showing an increase in GA3ox1 mRNA accumulation in response to DEX treatment compared with UBQ10 mRNA as internal control (n = 3). Error bars represent standard deviation (SD). (C) ChIP showing the direct interaction of IND-GR with the GA3ox1 gene. DNA obtained from pull-down with the GR antibody has been analyzed by qPCR using specific primers for the GA3ox1 gene. Values correspond to the ratios between pull-down and input DNA, both initially normalized to the Mu-like transposon. Values for four biological repeats are represented. P-value, <0.01. (D) β-Glucuronidase expression of AtGA3ox1 TC-GUS in young fruit (stage 15 according to Smyth et al. 1990) in the wild type (WT) and ind-1 mutant (ind-1). Bar, 500 μm. (E) β-Glucuronidase expression of AtGA3ox1 TC-GUS in young fruit (stage 15) cross-sections in th wild type and ind-1 mutant (ind-1). Bar, 100 μm.

Figure 2.

Low GA levels disrupt valve margin development and inhibit fruit opening. (A) SEM images showing the base of Arabidopsis fruit (stage 17b) of the wild type (WT), ind-1, ga4-1, and pIND >> GA2ox. Bar, 50 μm. (B) SEM images showing a close-up of valve margin and replum tissues. Pictures were taken in the middle of an Arabidopsis fruit (stage 17b) of the wild type, ind-1, ga4-1, and pIND >> GA2ox. Bar, 50 μm. (C) Shattering quantification of wild-type, ind-1, ga4-1, and pIND >> GA2ox fruits. Values represent the mean of three biological repeats (n = 3) (20 siliques each). Error bars represent standard deviation (SD).

Figure 3.

Low GA levels cause SL defects that resemble those of the alc mutant. (A) Diagram (based on a mPS-PI/confocal image) of the different cell layers in the wild-type valve margins: SL (orange) and LL (red). (B,C) TEM images showing valve margin tissues (cross-section) of the wild type (WT) (B) and ga4-1 mutant (C). SL cells are marked with asterisks (*). (R) Replum; (V) valve. Bar, 10 μm. (D) Quantification of cell sizes in the cell layer corresponding to the SL in the wild type (n = 38), alc-1 (n = 46), ga4-1 (n = 41), pIND >> GA2ox (n = 34), and alc-1; ga4-1 double mutant (n = 49). Error bars represent standard deviation (SD).

Figure 4.

DELLA proteins interact with ALC and inhibit valve margin development in the absence of GA. (A) Yeast two-hybrid interaction using ALC as a bait and GAI, RGA, and RGL2 as prey. (Top and bottom panels) Dilution series plated on selective medium (−Leu Trp His Ade) and control medium (−Leu Trp), respectively. (B) Projections of confocal sections showing expression of pRGA∷GFP-RGA in medial tissues of stage 15 gynoecium: untreated (Ø), treated with 1 μM PAC (+PAC), or treated with 10 μM gibberellin (+GA₃) for 24 h. (C) SEM images showing the base of a stage 17b fruit of the ga1-3 mutant treated with 1 μM GA₃ (left panel) or 100 μM GA₃ (right panel), and of the ga-3; rga-t2; gai-t6 triple mutant (middle panel). Bar = 100 μm. (D) Model of DELLA-mediated control of ALC by IND. IND induces GA biosynthesis via the direct activation of GA3ox1. Without GAs, ALC interacts with DELLA proteins. During valve margin development, IND-induced local GA production leads to DELLA degradation and release of ALC, which then controls SL genes.