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. 2011 Mar;23(3):923-41.
doi: 10.1105/tpc.110.081273. Epub 2011 Mar 11.

Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening

Rumyana Karlova  1 , Faye M Rosin, Jacqueline Busscher-Lange, Violeta Parapunova, Phuc T Do, Alisdair R Fernie, Paul D Fraser, Charles Baxter, Gerco C Angenent, Ruud A de Maagd
Affiliations

Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening

Rumyana Karlova et al. Plant Cell. 2011 Mar.

Abstract

Fruit ripening in tomato (Solanum lycopersicum) requires the coordination of both developmental cues as well as the plant hormone ethylene. Although the role of ethylene in mediating climacteric ripening has been established, knowledge regarding the developmental regulators that modulate the involvement of ethylene in tomato fruit ripening is still lacking. Here, we show that the tomato APETALA2a (AP2a) transcription factor regulates fruit ripening via regulation of ethylene biosynthesis and signaling. RNA interference (RNAi)-mediated repression of AP2a resulted in alterations in fruit shape, orange ripe fruits, and altered carotenoid accumulation. Microarray expression analyses of the ripe AP2 RNAi fruits showed altered expression of genes involved in various metabolic pathways, such as the phenylpropanoid and carotenoid pathways, as well as in hormone synthesis and perception. Genes involved in chromoplast differentiation and other ripening-associated processes were also differentially expressed, but softening and ethylene biosynthesis occurred in the transgenic plants. Ripening regulators RIPENING-INHIBITOR, NON-RIPENING, and COLORLESS NON-RIPENING (CNR) function upstream of AP2a and positively regulate its expression. In the pericarp of AP2 RNAi fruits, mRNA levels of CNR were elevated, indicating that AP2a and CNR are part of a negative feedback loop in the regulation of ripening. Moreover, we demonstrated that CNR binds to the promoter of AP2a in vitro.

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Figures

Figure 1.
Figure 1.
Phylogenetic Analysis of AP2a and Its Homologs and Comparison of Their Expression Pattern in Different Wild-Type Tissues and at Various Fruit Developmental Stages. (A) Protein phylogenetic tree of tomato AP2s and selected homologs indicate that five AP2 paralogs exist in tomato. Numbers at tree nodes indicate bootstrap values from a total of 1000 trials. (B) Relative expression profiles of AP2a, AP2b, AP2c, AP2d, and AP2e in different cv Moneymaker tissues obtained by quantitative RT-PCR. 1, Seedlings; 2, leaves; 3, roots; 4, flowers; 5, 5-mm fruits; 6, 1-cm fruits; 7, early green fruits; 8, mature green fruits, 9, breaker; 10, turning; 11-Br+7, 7 d after breaker stage, red fruits. Values represent means of two biological replicates, and vertical bars represent se of the means. (C) Relative expression profiles of Aa, AP2a; Ab, AP2b; Ac, AP2c; Ad, AP2d; and Ae, AP2e in sepals of AP2i-1 transgenic lines or the wild type (WT). Values represent means of two biological replicates, and vertical bars represent sd. The wild-type expression data are normalized to 1. Asterisks indicate P value < 0.05 (t test) when comparing data for each measurement between the AP2i and wild-type plants. (D) Relative expression profiles of Aa, AP2a; Ab, AP2b; and Ac, AP2c in the sepals of AP2i-2 transgenic lines or the wild type. Values represent means of two biological replicates, and vertical bars represent sd. The wild-type expression data are normalized to 1. Asterisk indicates P value < 0.05 (t test) when comparing data for each measurement between the AP2i and wild-type plants.
Figure 2.
Figure 2.
Ripening Effects of Decreased AP2a Expression. (A) Typical example of the ripening phenotype of AP2i-1 RNAi lines. Fully developed fruit (equivalent to Br + 7, 7 d after breaker stage) are orange in color, never achieving the full red color of the wild-type ripe tomatoes at the same stage. (B) Different stages of fruit ripening: (from left to right) mature green (~40 d postanthesis), turning, pink and red ripe (Br + 7) for wild-type (WT) and AP2i-1 RNAi lines. Fruits from independent transformant lines are shown (5, 8, 10, and 11, respectively), which are delayed in color development, never developing full red color. (C) Ripening wild-type fruits have an even, homogenous color and a smooth, intact surface during the orange and red stages of ripening. The color of the ripening AP2i-1 RNAi lines at the Br + 7 stage is not homogenous, with sectors displaying different colors. The fruit does not develop the red color but remains orange, even when the tissue has characteristics of being fully ripe or overripe (i.e., soft and mushy). The surface of the fruit splits open (arrows and in [A]). (D) Compared with the wild type (left), the fruit from AP2i-1 lines (right) at the mature green stage has a distorted shape, exhibiting strong indentations resulting in less round fruit. Additionally, the surface of the fruit is bumpy (arrows). (E) Wild-type (left) and AP2i-2-15 RNAi fruits (ap2-2-15) at Br + 7, showing ripening defects and uneven pigmentation.
Figure 3.
Figure 3.
AP2 RNAi Fruits Senescenced Faster Than the Wild-Type Fruits and Produced More Ethylene. (A) Comparison of wild-type (WT) and AP2i-1 fruits 14 d after harvesting at stage Br + 7 d. (B) Relative expression profiles of AP2a in mature green fruits, treated or not with ethephon. Values represent means of two biological replicates, and vertical bars represent sd. The wild-type expression data are normalized to 1. Asterisks indicate a significant P value < 0.05 (t test) when comparing data for each measurement between the treated and nontreated wild-type samples. (C) Fruits from Br and Br + 7 stages were sealed in airtight vials, and 2.5 mL of gas was sampled after 35 min. Values represent means of at least four fruits, and vertical bars represent sd. Asterisks indicate a significant P value < 0.05 (t test). [See online article for color version of this figure.]
Figure 4.
Figure 4.
Metabolic Comparison of the AP2i-1 and Wild-Type Fruits. (A) and (B) Comparison of the chlorophyll, carotenoid, and α-tocopherol levels (microgram/g fresh weight [FW]) in wild-type (wt) and transgenic AP2i-1 tomatoes at stage Br + 7. Biological replicates (11 fruits per sample) were performed in triplicate, and the data are presented as means ± sd. Student’s t test was used to determine significant differences between wild-type and transgenic fruits P < 0.01 (asterisks). (C) and (D) Comparison of the primary metabolites (amino acid and sugar) levels in wild-type and AP2i-1 tomatoes (six biological replicates) at stage Br + 7. Wild-type values are normalized to one. Vertical bars represent se of the means. Asterisks show statistically significant changes according to Student’s t test (P < 0.05). [See online article for color version of this figure.]
Figure 5.
Figure 5.
Relative Expression of AP2a in Fruits from Different Ripening-Defective Mutant Backgrounds. CNR protein binds directly to the promoter element of AP2a, and the two proteins colocalized in the nucleus of plant cells. (A) Relative expression profile of AP2a in wild-type cv WT-AC (Ailsa Craig), nor, rin, Gr, and Cnr fruits at Br +7 d, obtained by qRT-PCR. Values represent means of two biological replicates, and vertical bars represent sd. The wild-type expression data are normalized to 1. Asterisks indicate P value < 0.05 (t test). (B) SDS gel (left) showing GST-purified CNR protein used for the gel retardation assay. Main panel, gel retardation experiments with biotinylated DNA probes and GST-CNR purified protein. 1, AP2a promoter element containing the SBP true binding site; 2, AP2a promoter element, which does not contain the conserved SBP binding motif, used as a negative control. (C) Confocal scanning laser microscopy images of an Arabidopsis protoplast cotransfected with both CNR-YFP and AP2a-CFP constructs. Bar = 5 μm. (D) Upregulation of CNR in the pericarp of Br stage of the AP2i fruit compared with the wild-type cv Moneymaker (WT). The relative expression levels were obtained by qRT-PCR. Values represent the means of two biological replicates, and vertical bars represent sd. [See online article for color version of this figure.]
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