The regulation of MADS-box gene expression during ripening of banana and their regulatory interaction with ethylene

Abstract

Six MaMADS-box genes have been cloned from the banana fruit cultivar Grand Nain. The similarity of these genes to tomato LeRIN is low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns, specifically in fruit and the induction during ripening and in response to ethylene and 1-MCP, suggest that some of these genes may participate in ripening. MaMADS1, 2, and 3, are highly expressed in fruit only, while the others are expressed in fruit as well as in other organs. Moreover, the suites of MaMADS-box genes and their temporal expression differ in peel and pulp during ripening. In the pulp, the increase in MaMADS2, 3, 4, and 5 expression preceded an increase in ethylene production, but coincides with the CO(2) peak. However, MaMADS1 expression in pulp coincided with ethylene production, but a massive increase in its expression occurred late during ripening, together with a second wave in the expression of MaMADS2, 3, and 4. In the peel, on the other hand, an increase in expression of MaMADS1, 3, and to a lesser degree also of MaMADS4 and 2 coincided with an increase in ethylene production. Except MaMADS3, which was induced by ethylene in pulp and peel, only MaMADS4, and 5 in pulp and MaMADS1 in peel were induced by ethylene. 1-MCP applied at the onset of the increase in ethylene production, increased the levels of MaMADS4 and MaMADS1 in pulp, while it decreased MaMADS1, 3, 4, and 5 in peel, suggesting that MaMADS4 and MaMADS1 are negatively controlled by ethylene at the onset of ethylene production only in pulp. Only MaMADS2 is neither induced by ethylene nor by 1-MCP, and it is expressed mainly in pulp. Our results suggest that two independent ripening programs are employed in pulp and peel which involve the activation of mainly MaMADS2, 4, and 5 and later on also MaMADS1 in pulp, and mainly MaMADS1, and 3 in peel. Hence, our results are consistent with MaMADS2, a SEP3 homologue, acting in the pulp upstream of the increase in ethylene production similarly to LeMADS-RIN.

Fig. 1.

Isolation of MaMADS-box genes from Cavendish banana (cultivar Grand Nain). (A) Putative proteins alignment of MaMADS1–6 sequences isolated from banana fruit and the similarity among them. The gene sequences were deposited in http://www.ncbi.nlm.nih.gov/BLAST as EU869307, EU869306, EU869308, EU869309, EU869310, and EU869311, respectively. (B) Phylogenetic analysis of MaMADS1–4 with other SEP3 clade genes. The analysis was performed on the full-lengths of the individual genes. The SEP3 motif for each of the genes is presented on the right. The genes included in this alignment are: AoAOM1 (AAQ83834), SISEP3 (BAD10945.1), LItuAGL9 (AAX15920.1), PaAGL9.1 (AAX15923.1), HcSEP1 (BAC80253.1), PsM6 (AAX69068), DcSEP1 (AAZ95252.1), AdMADS (CAA48859.1), SaMADSD (CAA69916.1), PsMADS (CAA11258.1), PhFBP26 (AAF19164.1), NsMADS3 (AAD39034.1), LeTM5 (AAP57413.1), EcAGL9 (AAX15918.1), DeCDM44 (AAO22982.1), BpMADS (CAB95648.1), DEFH200 (CAA64743.1), PtMADS6 (AAO49811.1), AtSEP (AAT46095.1), VvMADS4 (AAM21344.1), AmDEFH72 (CAA64742.1). (C) Phylogenetic analysis of MaMADS5 with other AG genes. The existence of AG motifs for all the genes is shown. The genes included in this alignment are: DtSEEDSTICK (AAY86365.1), DnMADS2 (ABQ08574.1), AvMADS (BAD83772.1), HoMADS1 (AAF08830.2). (D) Phylogenetic analysis of MaMADS6 with other PI genes. The existence of PI motifs for all the genes are shown. The genes included in this alignment are: AoPI (ABB92623.1), P9PI (AAV28175.1), PI10 (AAV28490.1), HPI2 (AAD22494.2), PI1 (ABG90945.1). Lines underneath the genes mark the genes isolated from banana. Stars and dots indicate identity and similarity, respectively.

Fig. 2.

Expression patterns of MaMADS genes in various plant organs. BC, bulb core; R, root; PS, pseudostem; YL, young leaf; MF, male flower; FF, female flower; BL, bract leaf; MF(O), male flower ovary; FF(O), female flower ovary; PUPE, pulp at climacteric peak; PEPE, peel at climacteric peak. Expression was determined by Q-RT- PCR as relative quantification. The specificity of the primers was determined for each of the genes (see Table 1, Materials and methods), and the expression of both ribosomal RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as reference genes. Results are of a representative experiment, and are an average of three repetitions ±SD. The values of relative quantification obtained were 10⁵ higher.

Fig. 3.

Description of ethylene and carbon dioxide production (A) and ripening parameters (B) in whole fruit. The parameters were determined in banana from the upper hand immediately following harvest and at consecutive days during storage at 20 °C and 75% RH. Ripening parameters determined were peel colour (H^o angle) and firmness (N).

Fig. 4.

Dynamic changes in MaMADS-box genes expression in pulp tissue during ripening. The expression was determined in fruit from the upper hand according to details described in Fig. 2. The increase in CO₂ production appeared 6 d after harvest (DAH) (broken arrow) and the ethylene peak in these fruits was detected by 10 DAH (full arrow). Results are of a representative experiment, and represent an average of three repetitions ±SD.

Fig. 5.

Dynamic changes in MaMADS-box genes expression in peel tissue during ripening. The expression was determined in fruit from the upper hand according to details described in Fig. 2. An increase in CO₂ production appeared 6 d after harvest (DAH) (broken arrow) and the ethylene peak in these fruits was detected at 10 DAH (full arrow). Results are of a representative experiment, and represent an average of three repetitions ±SD.

Fig. 6.

Response to exogenous ethylene of MaMADS-box genes in peel and pulp. Banana fruits were treated with 10 μl l⁻¹ ethylene 4 d after harvest for 18 h. The expression was determined for control and ethylene-treated samples relative to other samples during fruit ripening and the results of day 5 after harvest are presented.

Fig. 7.

Response to 1-MCP of MaMADS-box genes in peel and pulp. Banana fruits were treated with 0.3 μl l⁻¹ 1-MCP at the onset of ethylene peak (8 d after harvest for 18 h). The expression was determined for control and 1-MCP-treated samples relative to other samples during fruit ripening and results of day 10 after harvest are presented.

Fig. 8.

Expression of MaMADS genes in rin/rin tomato fruit did not complement ripening. Ailsa Craig wild type, nearly isogenic rin/rin, and transgenic rin/rin tomato fruit were tagged at anthesis and designated as breaker stage at the same age as wild-type fruit which showed the first signs of colour change. Fruit were harvested 10 d post-breaker stage and photographed. Fruit are shown from (A) Ailsa Craig wt, (B) Ailsa Craig nearly isogenic for rin/rin, (C) rin/rin T₁ lines over-expressing MaMADS1 (line 15 on the left and line 18 on the right), (D) rin/rin T₁ lines over-expressing MaMADS2 (line 3 on the left and line 8 on the right). (This figure is available in colour at JXB online.)